Unit 2 discusses molecular tools for gene cloning, including restriction endonucleases, ligases, and other enzymes used in recombinant DNA technology. These enzymes are crucial for cutting DNA at specific points and joining DNA fragments together to create recombinant molecules. Restriction endonucleases cut DNA at specific recognition sequences and are classified into types based on their recognition sequences and cleavage mechanisms. Type II restriction endonucleases recognize short, specific DNA sequences and cut within these sequences, which is useful for precise and reproducible cutting of DNA required for cloning.
This document provides an introduction to genomics, proteomics, and comparative genomics. It discusses the central dogma of molecular biology involving DNA replication, transcription, and translation. It describes DNA and RNA structure and explains how genetic information flows from DNA to protein. The document also discusses genome sequencing, gene mapping, and how comparative analysis of genomes from different species can provide insights into evolutionary relationships and biological functions.
The document describes the dideoxy method of DNA sequencing. This method was foundational for modern genetic research as it provided the first ability to sequence DNA. It works by using dideoxy nucleotides that lack a hydroxyl group, preventing further nucleotide addition. DNA copies are made with each using one dideoxy nucleotide. After electrophoresis, the sequence is read by the length of DNA fragments terminated at each dideoxy position. While limited to short sequences, it enabled sequencing capabilities and projects like the Human Genome Project.
1. Artificial neural networks (ANNs) are being used as a bioinformatics approach for gene prediction and genetic diversity analysis. ANNs consist of interconnected layers that learn from input to output.
2. For gene prediction, a neural network is constructed with multiple input, hidden, and output layers. The input is a gene sequence and output is exon probability. Weights between layers are adjusted during training to recognize patterns.
3. ANNs have advantages over traditional statistical methods as they can model more complex data relationships without requiring detailed system information. Different ANN types exist for various applications in bioinformatics.
The document discusses several types and applications of polymerase chain reaction (PCR). It begins by explaining the basic three-step cycling process of PCR: denaturation, annealing of primers, and extension. It then describes several variations of PCR including inverse PCR, anchored PCR, asymmetric PCR, real-time PCR (RT-PCR), and PCR for site-directed mutagenesis. Inverse PCR is used to amplify unknown flanking genomic regions, while anchored and asymmetric PCR are used to generate single-stranded DNA products for downstream applications like sequencing. RT-PCR amplifies RNA sequences by first generating cDNA. PCR mutagenesis introduces mutations through altered primer sequences.
Molecular diagnostics is a collection of techniques used to analyse biological markers in the genome and proteome—the individual's genetic code and how their cells express their genes as proteins—by applying molecular biology to medical testing.
This document provides an introduction to genomics, proteomics, and comparative genomics. It discusses the central dogma of molecular biology involving DNA replication, transcription, and translation. It describes DNA and RNA structure and explains how genetic information flows from DNA to protein. The document also discusses genome sequencing, gene mapping, and how comparative analysis of genomes from different species can provide insights into evolutionary relationships and biological functions.
The document describes the dideoxy method of DNA sequencing. This method was foundational for modern genetic research as it provided the first ability to sequence DNA. It works by using dideoxy nucleotides that lack a hydroxyl group, preventing further nucleotide addition. DNA copies are made with each using one dideoxy nucleotide. After electrophoresis, the sequence is read by the length of DNA fragments terminated at each dideoxy position. While limited to short sequences, it enabled sequencing capabilities and projects like the Human Genome Project.
1. Artificial neural networks (ANNs) are being used as a bioinformatics approach for gene prediction and genetic diversity analysis. ANNs consist of interconnected layers that learn from input to output.
2. For gene prediction, a neural network is constructed with multiple input, hidden, and output layers. The input is a gene sequence and output is exon probability. Weights between layers are adjusted during training to recognize patterns.
3. ANNs have advantages over traditional statistical methods as they can model more complex data relationships without requiring detailed system information. Different ANN types exist for various applications in bioinformatics.
The document discusses several types and applications of polymerase chain reaction (PCR). It begins by explaining the basic three-step cycling process of PCR: denaturation, annealing of primers, and extension. It then describes several variations of PCR including inverse PCR, anchored PCR, asymmetric PCR, real-time PCR (RT-PCR), and PCR for site-directed mutagenesis. Inverse PCR is used to amplify unknown flanking genomic regions, while anchored and asymmetric PCR are used to generate single-stranded DNA products for downstream applications like sequencing. RT-PCR amplifies RNA sequences by first generating cDNA. PCR mutagenesis introduces mutations through altered primer sequences.
Molecular diagnostics is a collection of techniques used to analyse biological markers in the genome and proteome—the individual's genetic code and how their cells express their genes as proteins—by applying molecular biology to medical testing.
This document provides an overview of multiple sequence alignment (MSA). MSA is used to align biological sequences, such as DNA, RNA, or proteins, to find similarities and differences between sequences. The document outlines the goal of MSA as finding structural, functional, and evolutionary relationships. It describes the general considerations and steps of MSA, including pairwise comparison of sequences, cluster analysis to generate a hierarchy, and progressive alignment. Common MSA software and applications are also summarized.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Microarrays allow researchers to analyze gene expression across thousands of genes simultaneously. DNA probes are arrayed on a small glass or nylon slide, and labeled mRNA from samples is hybridized to the probes. Fluorescent scanning detects which genes are expressed. Data analysis includes normalization, distance metrics, clustering, and visualization to group genes with similar expression profiles and identify patterns of co-regulated genes. Microarrays enable functional genomics studies of development, disease, response to drugs or environmental factors, and more.
1. Microbial genomics analyzes and compares the complete genetic material of microorganisms. It provides insights into microbial evolution, diversity, applications in biotechnology, and treatment of pathogens.
2. Key tools for studying whole microbial genomes include pulsed field gel electrophoresis, large insert cloning vectors, whole genome sequencing approaches, microarray hybridization, and genome annotation pipelines.
3. Sequencing the first free-living organism, Haemophilus influenzae, involved random sequencing of small and large insert libraries, followed by closing gaps using various methods to produce the complete genome.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Ribonucleic acid (RNA) can be isolated from plant tissue through several methods for downstream applications. The document describes procedures for RNA isolation using denaturing buffers, phenol-chloroform extraction, and CTAB and TRIzol-based methods. Precautions like maintaining an RNase-free environment and using DEPC-treated materials are important for obtaining high-quality RNA. The procedures involve homogenizing tissues, separating RNA from other cellular components, and precipitating and purifying the isolated RNA.
This document discusses methods for extracting and enriching mRNA from biological samples. It describes acid guanidinium thiocyanate-phenol-chlorform extraction and CsCl gradient purification for total RNA extraction. For eukaryotes, mRNA is enriched using oligo-dT hybridization to the poly-A tail. For prokaryotes which lack a poly-A tail, methods include rRNA capture using probes, degradation of already processed RNAs leaving mRNA, selective polyadenylation of mRNA, and antibody capture of specific mRNAs associated with proteins. Purified mRNA can be used for applications like transcriptome sequencing, gene expression profiling, and techniques like RT-PCR, microarrays, and northern blotting.
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
Fluorescent in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells by permitting labeled probes to interact with complementary sequences, allowing detection of specific sequences located on chromosomes. FISH has many applications in molecular biology and medical science, including gene mapping, diagnosis of chromosomal abnormalities, and studies of cellular structure and function. It can be used to detect aberrations in chromosome number as well as single locus targets through use of different types of probes.
This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.
''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This document describes an electrophoretic mobility shift assay (EMSA) presentation. EMSA is a technique used to study interactions between proteins and DNA. It works by detecting a reduction in electrophoretic mobility of DNA when bound to a protein through gel electrophoresis. The presentation aims to describe the basic principles of EMSA, highlight its methods, and discuss applications such as determining binding affinities and studying conformational changes in DNA upon protein binding.
Structural databases like PDB, CSD, and CATH contain 3D structural information of proteins, small molecules, and macromolecules determined through techniques like X-ray crystallography and NMR spectroscopy. These databases provide bibliographic data, atomic coordinates, and other details for each entry. PDB contains protein structures, CSD contains organic and metal-organic structures, and CATH classifies protein domains hierarchically. Structural databases have wide applications in structure prediction, analysis, mining, comparison, classification, structure refinement, and database annotation.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses various methods for ligating DNA fragments, including blunt end ligation, sticky end ligation using linkers or adaptors, and homopolymeric tailing. Blunt end ligation is less efficient than sticky end ligation. Linkers and adaptors are oligonucleotides used to create sticky ends for ligation, while homopolymeric tailing uses terminal transferase to add homopolymer tails to blunt ends before ligation. The goal is to efficiently join vector and insert DNA fragments for recombinant DNA construction.
TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
Enzymes involved in rDNA technology.pptxPoonam Patil
This document discusses the key enzymes involved in recombinant DNA technology. It describes how restriction enzymes cut DNA at specific recognition sites, and DNA ligases join cut DNA fragments back together. The document outlines the process of recombinant DNA technology, including generating DNA fragments, inserting them into cloning vectors, introducing the vectors into host cells, and expressing the gene of interest. It provides details on various restriction enzymes and DNA-modifying enzymes used in genetic engineering applications.
DNA manipulating enzymes include nucleases, ligases, polymerases and modifying enzymes. Restriction endonucleases are enzymes that cut DNA molecules at specific recognition sequences. They produce either blunt or sticky ends. Restriction endonucleases are classified as type I, II or III, with type II having widespread use in recombinant DNA technology by allowing precise cutting of DNA molecules at specific sites.
This document provides an overview of multiple sequence alignment (MSA). MSA is used to align biological sequences, such as DNA, RNA, or proteins, to find similarities and differences between sequences. The document outlines the goal of MSA as finding structural, functional, and evolutionary relationships. It describes the general considerations and steps of MSA, including pairwise comparison of sequences, cluster analysis to generate a hierarchy, and progressive alignment. Common MSA software and applications are also summarized.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Microarrays allow researchers to analyze gene expression across thousands of genes simultaneously. DNA probes are arrayed on a small glass or nylon slide, and labeled mRNA from samples is hybridized to the probes. Fluorescent scanning detects which genes are expressed. Data analysis includes normalization, distance metrics, clustering, and visualization to group genes with similar expression profiles and identify patterns of co-regulated genes. Microarrays enable functional genomics studies of development, disease, response to drugs or environmental factors, and more.
1. Microbial genomics analyzes and compares the complete genetic material of microorganisms. It provides insights into microbial evolution, diversity, applications in biotechnology, and treatment of pathogens.
2. Key tools for studying whole microbial genomes include pulsed field gel electrophoresis, large insert cloning vectors, whole genome sequencing approaches, microarray hybridization, and genome annotation pipelines.
3. Sequencing the first free-living organism, Haemophilus influenzae, involved random sequencing of small and large insert libraries, followed by closing gaps using various methods to produce the complete genome.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Ribonucleic acid (RNA) can be isolated from plant tissue through several methods for downstream applications. The document describes procedures for RNA isolation using denaturing buffers, phenol-chloroform extraction, and CTAB and TRIzol-based methods. Precautions like maintaining an RNase-free environment and using DEPC-treated materials are important for obtaining high-quality RNA. The procedures involve homogenizing tissues, separating RNA from other cellular components, and precipitating and purifying the isolated RNA.
This document discusses methods for extracting and enriching mRNA from biological samples. It describes acid guanidinium thiocyanate-phenol-chlorform extraction and CsCl gradient purification for total RNA extraction. For eukaryotes, mRNA is enriched using oligo-dT hybridization to the poly-A tail. For prokaryotes which lack a poly-A tail, methods include rRNA capture using probes, degradation of already processed RNAs leaving mRNA, selective polyadenylation of mRNA, and antibody capture of specific mRNAs associated with proteins. Purified mRNA can be used for applications like transcriptome sequencing, gene expression profiling, and techniques like RT-PCR, microarrays, and northern blotting.
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
Fluorescent in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells by permitting labeled probes to interact with complementary sequences, allowing detection of specific sequences located on chromosomes. FISH has many applications in molecular biology and medical science, including gene mapping, diagnosis of chromosomal abnormalities, and studies of cellular structure and function. It can be used to detect aberrations in chromosome number as well as single locus targets through use of different types of probes.
This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.
''Electrophoretic Mobility Shift Assay'' by KATE, Wisdom DeebekeWisdom Deebeke Kate
This document describes an electrophoretic mobility shift assay (EMSA) presentation. EMSA is a technique used to study interactions between proteins and DNA. It works by detecting a reduction in electrophoretic mobility of DNA when bound to a protein through gel electrophoresis. The presentation aims to describe the basic principles of EMSA, highlight its methods, and discuss applications such as determining binding affinities and studying conformational changes in DNA upon protein binding.
Structural databases like PDB, CSD, and CATH contain 3D structural information of proteins, small molecules, and macromolecules determined through techniques like X-ray crystallography and NMR spectroscopy. These databases provide bibliographic data, atomic coordinates, and other details for each entry. PDB contains protein structures, CSD contains organic and metal-organic structures, and CATH classifies protein domains hierarchically. Structural databases have wide applications in structure prediction, analysis, mining, comparison, classification, structure refinement, and database annotation.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses various methods for ligating DNA fragments, including blunt end ligation, sticky end ligation using linkers or adaptors, and homopolymeric tailing. Blunt end ligation is less efficient than sticky end ligation. Linkers and adaptors are oligonucleotides used to create sticky ends for ligation, while homopolymeric tailing uses terminal transferase to add homopolymer tails to blunt ends before ligation. The goal is to efficiently join vector and insert DNA fragments for recombinant DNA construction.
TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
Enzymes involved in rDNA technology.pptxPoonam Patil
This document discusses the key enzymes involved in recombinant DNA technology. It describes how restriction enzymes cut DNA at specific recognition sites, and DNA ligases join cut DNA fragments back together. The document outlines the process of recombinant DNA technology, including generating DNA fragments, inserting them into cloning vectors, introducing the vectors into host cells, and expressing the gene of interest. It provides details on various restriction enzymes and DNA-modifying enzymes used in genetic engineering applications.
DNA manipulating enzymes include nucleases, ligases, polymerases and modifying enzymes. Restriction endonucleases are enzymes that cut DNA molecules at specific recognition sequences. They produce either blunt or sticky ends. Restriction endonucleases are classified as type I, II or III, with type II having widespread use in recombinant DNA technology by allowing precise cutting of DNA molecules at specific sites.
DNA manipulative enzymes can be grouped into four classes: nucleases cut DNA, ligases join DNA, polymerases copy DNA, and modifying enzymes add or remove chemical groups from DNA. Restriction endonucleases are nucleases that cut DNA at specific recognition sequences and are important for gene cloning. They produce either blunt or sticky ends. DNA ligase joins DNA fragments together by forming phosphodiester bonds. Polymerases like Klenow fragment and Taq polymerase copy DNA. Modifying enzymes alter DNA through addition or removal of chemical groups from the DNA backbone. Together, these enzymes enable the precise cutting and joining of DNA required for gene cloning experiments.
Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. Over 3000 restriction enzymes have been identified that cut DNA in different ways, leaving either sticky or blunt ends. They are essential tools in biotechnology and genetic engineering as they allow scientists to cut and recombine DNA from different sources.
Restriction Endonuclease: The Molecular Scissor of DNA - By RIKI NATHRIKI NATH
restriction enducleases are called the molecular scissors of DNA. types of restriction enzymes, their structures, subunits, most importantly the use of Type II restriction endonuclease in recombinant technology, mechanism of enzyme action and their applications.
This document discusses restriction enzymes, which are important tools in genetic engineering and recombinant DNA technology. Restriction enzymes cut DNA at specific recognition sequences and are used to cut DNA into fragments that can then be recombined in new ways. The document provides details on the discovery and functions of various restriction enzymes as well as other enzymes used in genetic engineering such as DNA ligase, alkaline phosphatase, polynucleotide kinase, and reverse transcriptase. It also discusses the use of restriction enzymes to build the first synthetic bacterial genome.
Nucleases are enzymes that cut nucleic acids. There are two main types - endonucleases which cut within strands, and exonucleases which degrade strands from the ends. Restriction endonucleases cut DNA at specific recognition sequences. Key examples are EcoRI, HindIII, and BamHI. Ribonucleases degrade RNA and are divided into endo- and exoribonucleases. RNase A is a common example. Nucleases play important roles in DNA repair and RNA processing through their cleavage activities.
DNA was discovered in 1868 and carries genetic traits. It is a double-stranded molecule composed of nucleotides that code for inherited characteristics. DNA replication is the process where DNA copies itself during cell division. It occurs in three main steps - initiation, elongation, and proofreading. DNA recombination and mutation can result in changes to the DNA sequence, altering traits or introducing variation. Recombinant DNA techniques cut and join DNA from different sources to produce modified sequences.
Recombinant enzymes are used in recombinant DNA technology and include nucleases, ligases, polymerases, and DNA modifying enzymes. Restriction enzymes cut DNA at specific recognition sequences and can produce blunt or sticky ends. Ligases join DNA fragments back together. Methylases protect host DNA from restriction enzymes by adding methyl groups to recognition sites. Topoisomerases regulate DNA supercoiling through transient single or double strand breaks. DNA gyrase is a type II topoisomerase that introduces negative supercoils to relieve strain on unwinding DNA.
Restriction enzymes cut DNA fragments at specific sites, leaving sticky or blunt ends. DNA ligase then joins the ends of DNA fragments together. Recombinant DNA technology uses these enzymes to isolate a gene of interest, cut it and a vector DNA, and join them via ligation to produce recombinant DNA that can be introduced into a host for replication. This allows for genetic analysis and applications in medicine, agriculture, and industry.
in gene cloning technique the cutting of DNA is essential. With the help of restriction endonuclease, it has been done. It also describes the restriction digest of a DNA molecule.
This document discusses restriction enzymes, which are proteins produced by bacteria to protect their DNA from viruses. Restriction enzymes recognize specific DNA sequences and cut the DNA at those sites. There are four main types of restriction enzymes. Type II enzymes cut DNA at or near their recognition sequences and are commonly used in genetic engineering to cut and combine DNA from different sources. This allows scientists to create recombinant DNA by splicing genes from one organism into another, enabling applications like producing pharmaceuticals in bacteria.
This document discusses restriction enzymes, including their discovery, types, subunits, nomenclature, recognition sequences, properties, and applications. Restriction enzymes are bacterial enzymes that cut DNA at specific recognition sequences. There are three main types - Type I cut DNA randomly, Type II cut within or near their recognition sequences, and Type III cut nearby. They are used in gene cloning, protein expression, DNA manipulation, and studying DNA sequences.
restrictionendonucleases-170328152703.pdfNoor Muhammed
This document discusses restriction enzymes, including their discovery, types, subunits, nomenclature, recognition sequences, properties, and applications. Restriction enzymes are bacterial enzymes that cut DNA at specific recognition sequences. There are three main types - Type I cut DNA randomly far from their recognition site, Type II cut within or near their recognition site, and Type III cut in the vicinity of their site. They are used in gene cloning, protein expression, DNA manipulation, and studying DNA sequences.
Assignment on Recombinant DNA Technology and Gene TherapyDeepak Kumar
Assignment on Recombinant DNA Technology and Gene Therapy Basic principles of recombinant DNA technology-Restriction enzymes, various types of vectors, Applications of recombinant DNA technology. Gene therapy- Various types of gene transfer techniques, clinical applications and recent advances in gene therapy
Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They are found in bacteria and archaea and serve to defend the host genome from foreign DNA like viruses. There are four main types of restriction enzymes that differ in composition, cofactor requirements, target sequence, and cleavage site. Restriction enzymes are important tools in biotechnology for manipulating DNA through cutting and joining of fragments. They allow for processes like DNA cloning, mapping, and sequencing.
Recombinant DNA technology involves manipulating DNA sequences in the laboratory. DNA is isolated, cut with restriction enzymes, and joined with DNA ligase. The recombinant DNA is inserted into a cloning vector and introduced into a host cell. Cells containing the recombinant DNA are selected and amplified. This allows large quantities of identical DNA molecules to be produced for analysis, comparison, and other purposes. Common applications include understanding disease, producing therapeutic proteins, disease prevention through vaccines, diagnosis, and gene therapy.
Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
This document summarizes the principles of dairy production, processing, and marketing. It discusses the composition of milk, including proteins, lactose, lipids, minerals, vitamins, and enzymes. The biosynthesis of milk constituents from the mammary gland is described. Finally, it discusses the structural and physical properties of milk as an emulsion, colloid, and solution, and how processing can impact these properties. Factors that influence milk quality like milking interval and lactation stage are also summarized.
This document discusses the process of PCR-based cloning. It explains that PCR is used to amplify a DNA sequence of interest and add restriction enzyme sites to the ends to allow for cloning into a plasmid. It provides details on designing forward and reverse primers, including adding a leader sequence, restriction site, and hybridization sequence. The document provides an example of adding EcoRI and NotI sites to a gene of interest for cloning into a recipient plasmid. It discusses factors to consider when choosing restriction enzymes and provides the specific primer sequences designed for the example.
This document discusses various gene transfer techniques including physical, chemical, and biological methods. It focuses on biological methods such as bactofection and transduction using viruses. Bactofection involves using bacteria to deliver genes directly into cells, while transduction uses viruses to package and deliver genes. The document also discusses chemical methods like calcium phosphate and lipofection, as well as physical methods such as electroporation, microinjection, and particle bombardment to introduce DNA into host cells.
1. DNA or proteins are separated by gel electrophoresis.
2. The molecules are then transferred to a membrane through blotting.
3. For Southern blotting, DNA is detected using labeled probes that hybridize to complementary DNA sequences. For Western blotting, proteins are detected using primary and secondary antibodies that bind to the protein of interest.
4. These techniques are used for applications like identifying genes, detecting infectious diseases and genetic disorders, and forensic analysis.
This document provides an overview of the Naive Bayes classifier machine learning algorithm. It begins by introducing Bayesian methods and Bayes' theorem. It then explains the basic probability formulas used in Naive Bayes. The document demonstrates how to calculate the probability that a patient has cancer using Bayes' theorem. It describes how Naive Bayes finds the maximum a posteriori hypothesis and deals with issues like parameter estimation and conditional independence. Finally, it works through an example predicting whether to play tennis using a sample training dataset.
(1) Bayes' theorem provides a framework for updating probabilities based on new information or evidence.
(2) It allows calculating the conditional probability of a hypothesis being true given observed data by combining the prior probability of the hypothesis with the likelihood of making the observation under that hypothesis.
(3) Bayesian inference uses Bayes' theorem to update beliefs as new data becomes available, representing uncertainty about unknown parameters as a probability distribution that is continually refined as more evidence is accumulated.
This document provides an overview of Bayes' theorem and examples of how to apply it to calculate conditional probabilities. It begins with definitions of partition, conditional probability, and the law of total probability. It then presents an example showing how to use Bayes' theorem to calculate the probability of a successful sidewalk sale given the probability of rain. Another example calculates the probability of a fatal car accident involving a light truck. The document concludes by discussing how Bayes' theorem could help calculate conditional probabilities for a loan workout decision based on new information about a borrower's years of business experience.
Presented by The Global Peatlands Assessment: Mapping, Policy, and Action at GLF Peatlands 2024 - The Global Peatlands Assessment: Mapping, Policy, and Action
Evolving Lifecycles with High Resolution Site Characterization (HRSC) and 3-D...Joshua Orris
The incorporation of a 3DCSM and completion of HRSC provided a tool for enhanced, data-driven, decisions to support a change in remediation closure strategies. Currently, an approved pilot study has been obtained to shut-down the remediation systems (ISCO, P&T) and conduct a hydraulic study under non-pumping conditions. A separate micro-biological bench scale treatability study was competed that yielded positive results for an emerging innovative technology. As a result, a field pilot study has commenced with results expected in nine-twelve months. With the results of the hydraulic study, field pilot studies and an updated risk assessment leading site monitoring optimization cost lifecycle savings upwards of $15MM towards an alternatively evolved best available technology remediation closure strategy.
Epcon is One of the World's leading Manufacturing Companies.EpconLP
Epcon is One of the World's leading Manufacturing Companies. With over 4000 installations worldwide, EPCON has been pioneering new techniques since 1977 that have become industry standards now. Founded in 1977, Epcon has grown from a one-man operation to a global leader in developing and manufacturing innovative air pollution control technology and industrial heating equipment.
Improving the viability of probiotics by encapsulation methods for developmen...Open Access Research Paper
The popularity of functional foods among scientists and common people has been increasing day by day. Awareness and modernization make the consumer think better regarding food and nutrition. Now a day’s individual knows very well about the relation between food consumption and disease prevalence. Humans have a diversity of microbes in the gut that together form the gut microflora. Probiotics are the health-promoting live microbial cells improve host health through gut and brain connection and fighting against harmful bacteria. Bifidobacterium and Lactobacillus are the two bacterial genera which are considered to be probiotic. These good bacteria are facing challenges of viability. There are so many factors such as sensitivity to heat, pH, acidity, osmotic effect, mechanical shear, chemical components, freezing and storage time as well which affects the viability of probiotics in the dairy food matrix as well as in the gut. Multiple efforts have been done in the past and ongoing in present for these beneficial microbial population stability until their destination in the gut. One of a useful technique known as microencapsulation makes the probiotic effective in the diversified conditions and maintain these microbe’s community to the optimum level for achieving targeted benefits. Dairy products are found to be an ideal vehicle for probiotic incorporation. It has been seen that the encapsulated microbial cells show higher viability than the free cells in different processing and storage conditions as well as against bile salts in the gut. They make the food functional when incorporated, without affecting the product sensory characteristics.
Optimizing Post Remediation Groundwater Performance with Enhanced Microbiolog...Joshua Orris
Results of geophysics and pneumatic injection pilot tests during 2003 – 2007 yielded significant positive results for injection delivery design and contaminant mass treatment, resulting in permanent shut-down of an existing groundwater Pump & Treat system.
Accessible source areas were subsequently removed (2011) by soil excavation and treated with the placement of Emulsified Vegetable Oil EVO and zero-valent iron ZVI to accelerate treatment of impacted groundwater in overburden and weathered fractured bedrock. Post pilot test and post remediation groundwater monitoring has included analyses of CVOCs, organic fatty acids, dissolved gases and QuantArray® -Chlor to quantify key microorganisms (e.g., Dehalococcoides, Dehalobacter, etc.) and functional genes (e.g., vinyl chloride reductase, methane monooxygenase, etc.) to assess potential for reductive dechlorination and aerobic cometabolism of CVOCs.
In 2022, the first commercial application of MetaArray™ was performed at the site. MetaArray™ utilizes statistical analysis, such as principal component analysis and multivariate analysis to provide evidence that reductive dechlorination is active or even that it is slowing. This creates actionable data allowing users to save money by making important site management decisions earlier.
The results of the MetaArray™ analysis’ support vector machine (SVM) identified groundwater monitoring wells with a 80% confidence that were characterized as either Limited for Reductive Decholorination or had a High Reductive Reduction Dechlorination potential. The results of MetaArray™ will be used to further optimize the site’s post remediation monitoring program for monitored natural attenuation.
Kinetic studies on malachite green dye adsorption from aqueous solutions by A...Open Access Research Paper
Water polluted by dyestuffs compounds is a global threat to health and the environment; accordingly, we prepared a green novel sorbent chemical and Physical system from an algae, chitosan and chitosan nanoparticle and impregnated with algae with chitosan nanocomposite for the sorption of Malachite green dye from water. The algae with chitosan nanocomposite by a simple method and used as a recyclable and effective adsorbent for the removal of malachite green dye from aqueous solutions. Algae, chitosan, chitosan nanoparticle and algae with chitosan nanocomposite were characterized using different physicochemical methods. The functional groups and chemical compounds found in algae, chitosan, chitosan algae, chitosan nanoparticle, and chitosan nanoparticle with algae were identified using FTIR, SEM, and TGADTA/DTG techniques. The optimal adsorption conditions, different dosages, pH and Temperature the amount of algae with chitosan nanocomposite were determined. At optimized conditions and the batch equilibrium studies more than 99% of the dye was removed. The adsorption process data matched well kinetics showed that the reaction order for dye varied with pseudo-first order and pseudo-second order. Furthermore, the maximum adsorption capacity of the algae with chitosan nanocomposite toward malachite green dye reached as high as 15.5mg/g, respectively. Finally, multiple times reusing of algae with chitosan nanocomposite and removing dye from a real wastewater has made it a promising and attractive option for further practical applications.
Recycling and Disposal on SWM Raymond Einyu pptxRayLetai1
Increasing urbanization, rural–urban migration, rising standards of living, and rapid development associated with population growth have resulted in increased solid waste generation by industrial, domestic and other activities in Nairobi City. It has been noted in other contexts too that increasing population, changing consumption patterns, economic development, changing income, urbanization and industrialization all contribute to the increased generation of waste.
With the increasing urban population in Kenya, which is estimated to be growing at a rate higher than that of the country’s general population, waste generation and management is already a major challenge. The industrialization and urbanization process in the country, dominated by one major city – Nairobi, which has around four times the population of the next largest urban centre (Mombasa) – has witnessed an exponential increase in the generation of solid waste. It is projected that by 2030, about 50 per cent of the Kenyan population will be urban.
Aim:
A healthy, safe, secure and sustainable solid waste management system fit for a world – class city.
Improve and protect the public health of Nairobi residents and visitors.
Ecological health, diversity and productivity and maximize resource recovery through the participatory approach.
Goals:
Build awareness and capacity for source separation as essential components of sustainable waste management.
Build new environmentally sound infrastructure and systems for safe disposal of residual waste and replacing current dumpsites which should be commissioned.
Current solid waste management situation:
The status.
Solid waste generation rate is at 2240 tones / day
collection efficiently is at about 50%.
Actors i.e. city authorities, CBO’s , private firms and self-disposal
Current SWM Situation in Nairobi City:
Solid waste generation – collection – dumping
Good Practices:
• Separation – recycling – marketing.
• Open dumpsite dandora dump site through public education on source separation of waste, of which the situation can be reversed.
• Nairobi is one of the C40 cities in this respect , various actors in the solid waste management space have adopted a variety of technologies to reduce short lived climate pollutants including source separation , recycling , marketing of the recycled products.
• Through the network, it should expect to benefit from expertise of the different actors in the network in terms of applicable technologies and practices in reducing the short-lived climate pollutants.
Good practices:
Despite the dismal collection of solid waste in Nairobi city, there are practices and activities of informal actors (CBOs, CBO-SACCOs and yard shop operators) and other formal industrial actors on solid waste collection, recycling and waste reduction.
Practices and activities of these actor groups are viewed as innovations with the potential to change the way solid waste is handled.
CHALLENGES:
• Resource Allocation.
Microbial characterisation and identification, and potability of River Kuywa ...Open Access Research Paper
Water contamination is one of the major causes of water borne diseases worldwide. In Kenya, approximately 43% of people lack access to potable water due to human contamination. River Kuywa water is currently experiencing contamination due to human activities. Its water is widely used for domestic, agricultural, industrial and recreational purposes. This study aimed at characterizing bacteria and fungi in river Kuywa water. Water samples were randomly collected from four sites of the river: site A (Matisi), site B (Ngwelo), site C (Nzoia water pump) and site D (Chalicha), during the dry season (January-March 2018) and wet season (April-July 2018) and were transported to Maseno University Microbiology and plant pathology laboratory for analysis. The characterization and identification of bacteria and fungi were carried out using standard microbiological techniques. Nine bacterial genera and three fungi were identified from Kuywa river water. Clostridium spp., Staphylococcus spp., Enterobacter spp., Streptococcus spp., E. coli, Klebsiella spp., Shigella spp., Proteus spp. and Salmonella spp. Fungi were Fusarium oxysporum, Aspergillus flavus complex and Penicillium species. Wet season recorded highest bacterial and fungal counts (6.61-7.66 and 3.83-6.75cfu/ml) respectively. The results indicated that the river Kuywa water is polluted and therefore unsafe for human consumption before treatment. It is therefore recommended that the communities to ensure that they boil water especially for drinking.
ENVIRONMENT~ Renewable Energy Sources and their future prospects.tiwarimanvi3129
This presentation is for us to know that how our Environment need Attention for protection of our natural resources which are depleted day by day that's why we need to take time and shift our attention to renewable energy sources instead of non-renewable sources which are better and Eco-friendly for our environment. these renewable energy sources are so helpful for our planet and for every living organism which depends on environment.
1. Unit 2
Molecular tools for gene cloning
Restriction and Modification systems: Restriction Endonucleases,
star activity of restriction enzymes, Methylases, Ligases.
Polynucleotide kinases, Phosphatases, DNA and RNA
polymerases, Reverse transcriptase, Terminal transferase,
DNAses (Extremophiles), Mung Bean Nuclease. RNases,
Topoisomerase.
2. Enzymes in rDNA technology
Once pure samples of DNA have been prepared, the next step in a gene cloning
experiment is construction of the recombinant DNA molecule (see Figure 1.1). To
produce this recombinant molecule, the vector, as well as the DNA to be cloned, must
be cut at specific points and then joined together in a controlled manner.
Cutting and joining are two examples of DNA manipulative techniques, a wide variety
of which have been developed over the past few years. As well as being cut and
joined, DNA molecules can be shortened, lengthened, copied into RNA or into new
DNA molecules, and modified by the addition or removal of specific chemical groups.
These manipulations, all of which can be carried out in the test tube, provide the
foundation not only for gene cloning, but also for studies of DNA biochemistry, gene
structure, and the control of gene expression.
Almost all DNA manipulative techniques make use of purified enzymes. Within the cell
these enzymes participate in essential processes such as DNA replication and
transcription, breakdown of unwanted or foreign DNA (e.g., invading virus DNA),
repair of mutated DNA, and recombination between different DNA molecules.
3. Enzymes in rDNA technology
After purification from cell extracts, many of these enzymes can
be persuaded to carry out their natural reactions, or something
closely related to them, under artificial conditions.
Although these enzymatic reactions are often straightforward,
most are absolutely impossible to perform by standard chemical
methods. Purified enzymes are therefore crucial to genetic
engineering and an important industry has sprung up around
their preparation, characterization, and marketing.
Commercial suppliers of high purity enzymes provide an essential
service to the molecular biologist. The cutting and joining
manipulations that underlie gene cloning are carried out by
enzymes called restriction endonucleases (for cutting) and
ligases (for joining).
4. The range of DNA manipulative
enzymes
DNA manipulative enzymes can be grouped into four broad
classes, depending on the type of reaction that they
catalyze:
Nucleases are enzymes that cut, shorten, or degrade nucleic
acid molecules.
Ligases join nucleic acid molecules together.
Polymerases make copies of molecules.
Modifying enzymes remove or add chemical groups.
5. Nucleases
Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link
one nucleotide to the next in a DNA strand. There are two different kinds of nuclease
(Figure 4.1):
Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
Endonucleases are able to break internal phosphodiester bonds within a DNA
molecule.
The main distinction between different exonucleases lies in the number of strands
that are degraded when a double-stranded molecule is attacked.
The enzyme called Bal31 (purified from the bacterium Alteromonas espejiana) is an
example of an exonuclease that removes nucleotides from both strands of a double-
stranded molecule (Figure 4.2a). The greater the length of time that Bal31 is allowed
to act on a group of DNA molecules, the shorter the resulting DNA fragments will be.
In contrast, enzymes such as E. coli exonuclease III has got 3’ to 5’ exonuclease activity.
(Figure 4.2b).
6.
7.
8. Nucleases
The same criterion can be used to classify endonucleases. S1
endonuclease (from the fungus Aspergillus oryzae) only cleaves
single strands (Figure 4.3a), whereas deoxyribonuclease I (DNase I),
which is prepared from cow pancreas, cuts both singleand
double-stranded molecules (Figure 4.3b).
DNase I is non-specific in that it attacks DNA at any internal
phosphodiester bond, so the end result of prolonged DNase I action is
a mixture of mononucleotides and very short oligonucleotides. On the
other hand, the special group of enzymes called restriction
endonucleases cleave doublestranded DNA only at a limited number of
specific recognition sites (Figure 4.3c).
9.
10. Enzymes for cutting DNA—restriction endonucleases
Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. This is
illustrated by the way in which the vector is cut during construction of a recombinant DNA molecule (Figure
4.7a).
Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be
inserted: a molecule that is cut more than once will be broken into two or more separate fragments and will
be of no use as a cloning vector.
Furthermore, each vector molecule must be cut at exactly the same position on the circle, random cleavage
is not satisfactory. It should be clear that a very special type of nuclease is needed to carry out this
manipulation. Often it is also necessary to cleave the DNA that is to be cloned (Figure 4.7b). There are two
reasons for this.
• First, if the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, then that gene will
have to be cut out of the large (often greater than 80 kb) DNA molecules
• Second, large DNA molecules may have to be broken down simply to produce fragments small enough
to be carried by the vector.
Most cloning vectors exhibit a preference for DNA fragments that fall into a particular size range: most
plasmid-based vectors, for example, are very inefficient at cloning DNA molecules more than 8 kb in length.
Purified restriction endonucleases allow the molecular biologist to cut DNA molecules in the precise,
reproducible manner required for gene cloning. The discovery of these enzymes, which led to Nobel Prizes
for W. Arber, H. Smith, and D. Nathans in 1978, was one of the key breakthroughs in the development of
genetic engineering.
11.
12. The discovery and function of restriction
endonucleases
The initial observation that led to the eventual discovery of restriction endonucleases was
made in the early 1950s, when it was shown that some strains of bacteria are immune to
bacteriophage infection, a phenomenon referred to as host-controlled restriction.
The mechanism of restriction is not very complicated, even though it took over 20 years to be
fully understood. Restriction occurs because the bacterium produces an enzyme that degrades
the phage DNA before it has time to replicate and direct synthesis of new phage particles
(Figure 4.8a).
The bacterium’s own DNA, the destruction of which would of course be lethal, is protected
from attack because it carries additional methyl groups that block the degradative enzyme
action (Figure 4.8b).
These degradative enzymes are called restriction endonucleases and are synthesized by many,
perhaps all, species of bacteria: over 2500 different ones have been isolated and more than
300 are available for use in the laboratory.
13.
14. Restriction Endonuclease
Nomenclature
Restriction endonucleases are named according to the organism in which
they were discovered, using a system of letters and numbers. For example,
HindIII was discovered in Haemophilus influenza (strain d). The Roman
numerals are used to identify specific enzymes from bacteria that contain
multiple restriction enzymes indicating the order in which restriction enzymes
were discovered in a particular strain.
15. Classification of Restriction
Endonucleases
There are three major classes of restriction
endonucleases based on the types of sequences
recognized, the nature of the cut made in the
DNA, and the enzyme structure:
• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes
16. Type I restriction enzymes
• These enzymes have both restriction and modification activities.
Restriction depends upon the methylation status of the target DNA.
• Cleavage occurs approximately 1000 bp away from the recognition site.
• The recognition site is asymmetrical and is composed of two specific
portions in which one portion contain 3–4 nucleotides while another
portion contain 4–5 nucleotides and both the parts are separated by a
non-specific spacer of about 6–8 nucleotides.
• They require S-adenosylmethionine (SAM), ATP, and magnesium ions
(Mg2+) for activity.
• These enzymes are composed of mainly three subunits, a specificity
subunit that determines the DNA recognition site, a restriction subunit,
and a modification subunit
17. Type III restriction enzymes
•These enzymes recognize and methylate the same DNA
sequence but cleave 24–26 bp away.
•They have two different subunits, in which one subunit (M)
is responsible for recognition and modification of DNA
sequence and other subunit (R) has nuclease action.
• Mg+2 ions, ATP are needed for DNA cleavage and process of
cleavage is stimulated by SAM.
•Cleave only one strand. Two recognition sites in opposite
orientation are necessary to break the DNA duplex
18. Type II restriction endonucleases cut DNA at specific
nucleotide sequences
The central feature of type II restriction endonucleases (which will be referred to simply as
“restriction endonucleases” from now on) is that each enzyme has a specific recognition
sequence at which it cuts a DNA molecule.
A particular enzyme cleaves DNA at the recognition sequence and nowhere else. For
example, the restriction endonuclease called PvuI (isolated from Proteus vulgaris) cuts DNA
only at the hexanucleotide CGATCG.
In contrast, a second enzyme from the same bacterium, called PvuII, cuts at a different
hexanucleotide, in this case CAGCTG.
Many restriction endonucleases recognize hexanucleotide target sites, but others cut at
four, five, eight, or even longer nucleotide sequences. Sau3A (from Staphylococcus aureus
strain 3A) recognizes GATC, and AluI (Arthrobacter luteus) cuts at AGCT.
There are also examples of restriction endonucleases with degenerate recognition
sequences, meaning that they cut DNA at any one of a family of related sites. HinfI
(Haemophilus influenzae strain Rf), for instance, recognizes GANTC, so cuts at GAATC,
GATTC, GAGTC, and GACTC.
19. The steps involved in DNA binding and cleavage
by a type II restriction endonuclease
• These enzymes have nonspecific contact with DNA and initially bind
to DNA as dimers.
• The target site is then located by a combination of linear diffusion or
“sliding” of the enzyme along the DNA over short distances, and
hopping/jumping over longer distances.
• Once the target restriction site is located, the recognition process
(coupling) triggers large conformational changes of the enzyme and the
DNA, which leads to activation of the catalytic center.
• Catalysis results in hydrolysis of phosphodiester bond and
product release.
22. Blunt ends and sticky ends
Blunt ends
The exact nature of the cut produced by a restriction endonuclease is of considerable
importance in the design of a gene cloning experiment. Many restriction endonucleases make a
simple double-stranded cut in the middle of the recognition sequence (Figure 4.9a), resulting in
a blunt end or flush end. PvuII and AluI are examples of blunt end cutters.
Sticky Ends
Other restriction endonucleases cut DNA in a slightly different way. With these enzymes, the
two DNA strands are not cut at exactly the same position. Instead the cleavage is staggered,
usually by two or four nucleotides, so that the resulting DNA fragments have short single-
stranded overhangs at each end (Figure 4.9b).
These are called sticky or cohesive ends, as base pairing between them can stick the DNA
molecule back together again. One important feature of sticky end enzymes is that restriction
endonucleases with different recognition sequences may produce the same sticky ends.
BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—both produce GATC
sticky ends (Figure 4.9c). The same sticky end is also produced by Sau3A, which recognizes only
the tetranucleotide GATC. Fragments of DNA produced by cleavage with either of these
enzymes can be joined to each other, as each fragment carries a complementary sticky end.
23.
24. Performing a restriction digest in the laboratory
As an example, we will consider how to digest a sample of λ DNA (concentration 125 mg/ml) with BglII.
First, the required amount of DNA must be pipetted into a test tube. The amount of DNA that will be
restricted depends on the nature of the experiment.
In this case we will digest 2 μg of λ DNA, which is contained in 16 μl of the sample (Figure 4.11a). Very
accurate micropipettes will therefore be needed. The other main component in the reaction will be the
restriction endonuclease, obtained from a commercial supplier as a pure solution of known concentration.
But before adding the enzyme, the solution containing the DNA must be adjusted to provide the correct
conditions to ensure maximal activity of the enzyme. Most restriction endonucleases function adequately
at pH 7.4, but different enzymes vary in their requirements for ionic strength (usually provided by sodium
chloride (NaCl)) and magnesium (Mg2+) concentration (all type II restriction endonucleases require Mg2+ in
order to function).
It is also advisable to add a reducing agent, such as dithiothreitol (DTT), which stabilizes the enzyme and
prevents its inactivation. Providing the right conditions for the enzyme is very important—incorrect NaCl or
Mg2+ concentrations not only decrease the activity of the restriction endonuclease, they might also cause
changes in the specificity of the enzyme, so that DNA cleavage occurs at additional, non-standard
recognition sequences.
After adding suitable composition of the buffer, The restriction endonuclease can now be added.
By convention, 1 unit of enzyme is defined as the quantity needed to cut 1 μg of DNA in 1 hour, so we
need 2 units of BglII to cut 2 μg of λ DNA. BglII is frequently obtained at a concentration of 4 units/ μl, so 0.5
μl is sufficient to cleave the DNA.
25. The final ingredients in the reaction mixture are therefore 0.5 μl BglII + 2μl of BglII
buffer+ 1.5 μl water + 16 μl of template DNA giving a final volume of 20 μl (Figure
4.11c).
The last factor to consider is incubation temperature. Most restriction endonucleases,
including BglII, work best at 37°C, but a few have different requirements.
TaqI, for example, is a restriction enzyme from Thermus aquaticus and, like Taq DNA
polymerase, has a high working temperature. Restriction digests with TaqI must be
incubated at 65°C to obtain maximum enzyme activity.
After 1 hour the restriction should be complete (Figure 4.11d). If the DNA fragments
produced by restriction are to be used in cloning experiments, the enzyme must
somehow be destroyed so that it does not accidentally digest other DNA molecules
that may be added at a later stage. There are several ways of “killing” the enzyme. For
many a short incubation at 70°C is sufficient, for others phenol extraction or the
addition of ethylenediamine tetraacetate (EDTA), which binds Mg2+ ions preventing
restriction endonuclease action, is used (Figure 4.11e).
26. Applications of REs
In various applications related to genetic engineering DNA is
cleaved by using restriction enzymes.
•They are used in the process of insertion of genes into plasmid
vectors during gene cloning and protein expression experiments.
•Restriction enzymes can also be used to distinguish gene alleles by
specifically recognizing single base changes in DNA known as single
nucleotide polymorphisms (SNPs). This is only possible if a
mutation alters the restriction site present in the allele.
•Restriction enzymes are used for Restriction Fragment Length
Polymorphism (RFLP) analysis for identifying individuals or strains of
a particular species.
27. Ligases – joins DNA molecules together
DNA ligase catalyses the formation of phosphodiester bond between two deoxynucleotide residues of
two DNA strands.
DNA ligase enzyme requires a free hydroxyl group at the 3´ -end of one DNA chain and a phosphate group
at the 5´-end of the other and requires energy in the process.
E.coli and other bacterial DNA ligase utilizes NAD+ as energy donor, whereas in T4 bacteriophage, T4 DNA
ligase uses ATP as cofactor.
The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA after lagging strand
formation to join the okazaki fragments. This joining process is essential for the normal synthesis of DNA
and for repairing damaged DNA. It has been exploited by genetic engineers to join DNA chains to form
recombinant DNA molecules. Usually single stranded break are repaired using the complimentary strand
as the template but sometimes double stranded breaks can also be repaired with the help of DNA
ligase IV.
The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs ATP as a
cofactor. The enzyme from E. coli uses cofactor NAD. Except this, the catalysis mechanism is somewhat
similar for both the ligases. The role of cofactor is splitting and forming an enzyme-AMP complex which
further aids in formation of phosphodiester bonds between hydroxyl and phosphate groups by exposing
them.
28. Ligases are the most commonly used enzymes for
carrying out ligations. They are part of the routine battery
of enzymes required by a cell for the maintenance of its
DNA. They are used in joining together adjacent Okazaki
fragments produced in replication, and in sealing the nicks
that arise from damage and repair processes.
T4 DNA ligase is encoded by bacteriophage T4, and is
produced on infection of E. coli cells. It can carry out both
blunt-ended and sticky-ended ligations, and requires ATP.
It requires a 3’-hydroxyl and a 5’-phosphate group on the
molecules to be joined.
29. E. coli DNA ligase is the endogenous bacterial enzyme.
Unlike T4 DNA ligase, it is unable to carry out
blunt-ended ligations (or does so only very inefficiently)
and, therefore, is particularly useful if such ligations need
to be avoided.
This might be the case if you were trying to seal nicks in
damaged DNA without also joining non-contiguous
sequences. Like the T4 ligase, E. coli DNA ligase requires
a 3’-hydroxyl and a 5’-phosphate group, but it requires
nicotinamide adenine dinucleotide (NAD+) as a cofactor.
Essentially the same reaction is catalysed by each of
these ligases
30. In both cases, adenosine monophosphate (AMP) is added to the
5’-phosphate, of one DNA molecule liberating either pyrophosphate
from ATP or nicotinamide mononucleotide from NAD+
. The AMP is
then displaced in a nucleophilic attack by the 3’-hydroxyl of the
other DNA molecule.
Mechanism of ligation by T4DNA ligase (using ATP)or E.coli ligase (using NAD+).
31. Mechanism of Action of DNA Ligases
•ATP, or NAD+, reacts with the ligase enzyme to form a covalent enzyme–AMP complex
in which the AMP is linked to ε-amino group of a lysine residue in the active site of the
enzyme through a phospho-amide bond.
• The AMP moiety activates the phosphate group at the 5´-end of the DNA molecule to
be joined. It is called as the donor.
•The final step is a nucleophilic attack by the 3´-hydroxyl group on this activated
phosphorus atom which acts as the acceptor. A phosphodiester bond is formed and
AMP is released.
•The reaction is driven by the hydrolysis of the pyrophosphate released during the
formation of the enzyme–adenylate complex. Two high-energy phosphate bonds are
spent in forming a phosphodiester bond in the DNA backbone with ATP serving as energy
source.
•The temperature optimum for T4 DNA ligase mediated ligation in vitro is 16˚C. However
ligation is also achieved by incubation at 4˚C by incubating over night or at room
temperature condition by incubating for 30 minutes.
•Adenylate and DNA-adenylate are the important intermediates of the phosphodiester
bond forming pathway.
33. Applications of DNA
ligase
• DNA ligase enzyme is used by cells to join the “okazaki fragments”
during DNA replication process. In molecular cloning, ligase
enzyme has been routinely used to construct a recombinant DNA.
• Joining of adapters and linkers to blunt end DNA molecule.
• Cloning of restricted DNA to vector to construct recombinant vector.
Ligation of a gene fragment into the vector and transformation of the cell.
34. Use of DNA ligase to create a covalent DNA recombinant
joined through association of termini generated by EcoRI.
35. Application of alkaline phosphatase treatment to prevent
recircularization of vector plasmid without insertion of foreign
DNA.
36. Sticky ends increase the efficiency of ligation
The ligation reaction carried out by two blunt-ended fragments is not very
efficient. This is because the ligase is unable to “catch hold” of the
molecule to be ligated, and has to wait for chance associations to bring the
ends together.
If possible, blunt end ligation should be performed at high DNA
concentrations, to increase the chances of the ends of the molecules
coming together in the correct way.
In contrast, ligation of complementary sticky ends is much more efficient.
This is because compatible sticky ends can base pair with one another by
hydrogen bonding (Figure 4.20b), forming a relatively stable structure for
the enzyme to work on. If the phosphodiester bonds are not synthesized
fairly quickly then the sticky ends fall apart again. These transient, base-
paired structures do, however, increase the efficiency of ligation by
increasing the length of time the ends are in contact with one another.
38. Putting sticky ends onto a blunt-ended molecule
For the reasons detailed in the preceding section, compatible
sticky ends are desirable on the DNA molecules to be ligated
together in a gene cloning experiment. Often these sticky
ends can be provided by digesting both the vector and the
DNA to be cloned with the same restriction endonuclease, or
with different enzymes that produce the same sticky end,
but it is not always possible to do this.
A common situation is where the vector molecule has
sticky ends, but the DNA fragments to be cloned are
blunt-ended. Under these circumstances one of three
following methods can be used to put the correct sticky
ends onto the DNA fragments.
39. Linkers
The first of these methods involves the use of linkers. These are short pieces of double
stranded DNA, of known nucleotide sequence, that are synthesized in the test tube.
A typical linker is shown in Figure 4.21a.
It is blunt-ended, but contains a restriction site, BamHI in the example shown. DNA ligase can
attach linkers to the ends of larger blunt ended DNA molecules. Although a blunt end
ligation, this particular reaction can be performed very efficiently because synthetic
oligonucleotides, such as linkers, can be made in very large amounts and added into the
ligation mixture at a high concentration.
More than one linker will attach to each end of the DNA molecule, producing the chain
structure shown in Figure 4.21b. However, digestion with BamHI cleaves the chains at the
recognition sequences, producing a large number of cleaved linkers and the original DNA
fragment, now carrying BamHI sticky ends. This modified fragment is ready for ligation into a
cloning vector restricted with BamHI.
40.
41. A decameric linker molecule containing an EcoRI target site is joined by T4 DNA
ligase to both ends of flush ended foreign DNA. Cohesive ends are then
generated by EcoRI. This DNA can then be incorporated into a vector that has
been treated with the same restriction endonuclease
42. Adaptors
There is one potential drawback with the use of linkers. Consider what would happen if the
blunt-ended molecule shown in Figure 4.21b contained one or more BamHI recognition
sequences. If this was the case, the restriction step needed to cleave the linkers and produce
the sticky ends would also cleave the blunt-ended molecule (Figure 4.22). The resulting
fragments will have the correct sticky ends, but that is no consolation if the gene contained
in the blunt-ended fragment has now been broken into pieces.
The second method of attaching sticky ends to a blunt-ended molecule is designed to avoid
this problem. Adaptors, like linkers, are short synthetic oligonucleotides. But unlike linkers,
an adaptor is synthesized so that it already has one sticky end (Figure 4.23a). The idea is of
course to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to
produce a new molecule with sticky ends. This may appear to be a simple method but in
practice a new problem arises. The sticky ends of individual adaptor molecules could base
pair with each other to form dimers (Figure 4.23b), so that the new DNA molecule is still
blunt-ended (Figure 4.23c). The sticky ends could be recreated by digestion with a restriction
endonuclease, but that would defeat the purpose of using adaptors in the first place.
43. The problem can be resolved by synthesizing Adaptor molecules such that the
blunt end is the same as “natural” DNA, but the sticky end is different. The 3ʹ-OH
terminus of the sticky end is the same as usual, but the 5ʹ-P terminus is modified:
it lacks the phosphate group, and is in fact a 5ʹ-OH terminus (Figure 4.25a). DNA
ligase is unable to form a phosphodiester bridge between 5ʹ-OH and 3ʹ-OH ends.
The result is that, although base pairing is always occurring between the sticky
ends of adaptor molecules, the association is never stabilized by ligation (Figure
4.25b). Adaptors can therefore be ligated to a blunt-ended DNA molecule but not
to themselves. After the adaptors have been attached, the abnormal 5ʹ-OH
terminus is converted to the natural 5ʹ-P form by treatment with the enzyme
polynucleotide kinase (p. 50), producing a sticky-ended fragment that can be
inserted into an appropriate vector.
44. Figure 4.25 The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5ʹ-OH
terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors.
45. Use of a BamHI adaptor molecule. A synthetic adaptor molecule is ligated to
the foreign DNA. The adaptor is used in the 5′-hydroxyl form to prevent
self-polymerization. The foreign DNA plus ligated adaptors is phosphorylated
at the 5′-termini and ligated into the vector previously cut with BamHI.
46. Producing sticky ends by homopolymer tailing
The technique of homopolymer tailing offers a radically different approach to the
production of sticky ends on a blunt-ended DNA molecule.
A homopolymer is simply a polymer in which all the subunits are the same. A DNA
strand made up entirely of, say, deoxyguanosine is an example of a homopolymer, and
is referred to as polydeoxyguanosine or poly(dG). Tailing involves using the enzyme
terminal deoxynucleotidyl transferase (p. 50) to add a series of nucleotides onto the
3ʹ-OH termini of a double-stranded DNA molecule.
If this reaction is carried out in the presence of just one deoxyribonucleotide, a
homopolymer tail is produced (Figure 4.26a). Of course, to be able to ligate together
two tailed molecules, the homopolymers must be complementary.
Frequently polydeoxycytosine (poly(dC)) tails are attached to the vector and poly(dG)
to the DNA to be cloned. Base pairing between the two occurs when the DNA
molecules are mixed (Figure 4.26b).
In practice, the poly(dG) and poly(dC) tails are not usually exactly the same length,
and the base-paired recombinant molecules that result have nicks as well as
discontinuities (Figure 4.26c).
47. Repair is therefore a two-step process,
using Klenow polymerase to fill in the
nicks followed by DNA ligase to
synthesize the final phosphodiester
bonds. This repair reaction does not
always have to be performed in the test
tube. If the complementary
homopolymer tails are longer than
about 20 nucleotides, then quite stable
base-paired associations are formed.
A recombinant DNA molecule, held
together by base pairing although not
completely ligated, is often stable
enough to be introduced into the host
cell in the next stage of the cloning
experiment. Once inside the host, the
cell’s own DNA polymerase and DNA
ligase repair the recombinant DNA
molecule, completing the construction
begun in the test tube.
48. Use of calf-thymus terminal deoxynucleotidyltransferase to add
complementary homopolymer tails to two DNA molecules. Use
of calf-thymus terminal deoxynucleotidyltransferase to add
complementary homopolymer tails to two DNA molecules.
49. Blunt end ligation with a DNA topoisomerase
A more sophisticated, but easier and generally more efficient way of carrying out blunt end ligation,
is to use a special type of enzyme called a DNA topoisomerase. Topoisomerase is an enzyme that
introduces or removes turns from the double helix by breakage and reunion of one or both
polynucleotides.
In the cell, DNA topoisomerases are involved in processes that require turns of the double helix to be
removed or added to a double-stranded DNA molecule. Turns are removed during DNA replication in
order to unwind the helix and enable each polynucleotide to be replicated, and are added to newly
synthesized circular molecules to introduce supercoiling. DNA topoisomerases are able to
separate the two strands of a DNA molecule without actually rotating the double helix. They achieve
this feat by causing transient single- or double-stranded breakages in the DNA backbone (Figure
4.27). DNA topoisomerases therefore have both nuclease and ligase activities.
To carry out blunt end ligation with a topoisomerase, a special type of cloning vector is needed. This
is a plasmid that has been linearized by the nuclease activity of the DNA topoisomerase enzyme
from vaccinia virus. The vaccinia topoisomerase cuts DNA at the sequence CCCTT, which is present
just once in the plasmid. After cutting the plasmid, topoisomerase enzymes remain covalently bound
to the resulting blunt ends. The reaction can be stopped at this point, enabling the vector to be
stored until it is needed.
Cleavage by the topoisomerase results in 5ʹ-OH and 3ʹ-P termini (Figure 4.28a). If the blunt-ended
molecules to be cloned have been produced from a larger molecule by cutting with a restriction
enzyme, then they will have 5ʹ-P and 3ʹ-OH ends. Before mixing these molecules with the vector,
their terminal phosphates must be removed to give 5ʹ-OH ends that can ligate to the 3ʹ-P termini of
the vector. The molecules are therefore treated with alkaline phosphatase (Figure 4.28b).
50. Figure 4.27
The mode of action of a Type 1 DNA
topoisomerase, which removes or adds turns
to a double helix by making a transient break
in one of the strands.
Adding the phosphatased molecules to the
vector reactivates the bound
topoisomerases, which proceed to the
ligation phase of their reaction. Ligation
occurs between the 3ʹ-P ends of the
vectors and the 5ʹ-OH ends of the
phosphatased molecules.
The blunt-ended molecules therefore
become inserted into the vectors. Only one
strand is ligated at each junction point
(Figure 4.28c), but this is not a problem
because the discontinuities will be repaired
by cellular enzymes after the recombinant
molecules have been introduced into the
host bacteria.
51. Figure 4.28 Blunt end ligation with a DNA topoisomerase. (a) Cleavage of the vector with the
topoisomerase leaves blunt ends with 5ʹ-OH and 3ʹ-P termini. (b) The molecule to be cloned
must therefore be treated with alkaline phosphatase to convert its 5ʹ-P ends into 5ʹ-OH
termini. (c) The topoisomerase ligates the 3ʹ-P and 5ʹ-OH ends, creating a double-stranded
molecule with two discontinuities, which are repaired by cellular enzymes after introduction
into the host bacteria.
52. DNA polymerases
DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an
existing DNA or RNA template (Figure 4.5a). Most polymerases can function only if the
template possesses a double-stranded region that acts as a primer for initiation of
polymerization
DNA polymerase I, which is usually prepared from E. coli. This enzyme attaches to a short
single-stranded region (or nick) in a mainly double-stranded DNA molecule, and then
synthesizes a completely new strand, degrading the existing strand as it proceeds (Figure
4.5b). DNA polymerase I is therefore an example of an enzyme with a dual activity—DNA
polymerization and DNA degradation
The polymerase and nuclease activities of DNA polymerase I are controlled by different parts
of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the
polypeptide, so removal of this segment leaves a modified enzyme that retains the
polymerase function but is unable to degrade DNA. This modified enzyme, called the Klenow
fragment, can still synthesize a complementary DNA strand on a single-stranded template,
but as it has no nuclease activity it cannot continue the synthesis once the nick is filled in
(Figure 4.5c).
Several other enzymes—natural polymerases and modified versions—have similar properties
to the Klenow fragment. The major application of these polymerases is in DNA sequencing.
53. The Taq DNA polymerase used in the polymerase chain reaction
(PCR) is the DNA polymerase I enzyme of the bacterium
Thermus aquaticus. This organism lives in hot springs, and
many of its enzymes, including the Taq DNA polymerase, are
thermostable, meaning that they are resistant to denaturation
by heat treatment. This is the special feature of Taq DNA
polymerase that makes it suitable for PCR, because if it was not
thermostable it would be inactivated when the temperature of
the reaction is raised to 94°C to denature the DNA.
The final type of DNA polymerase that is important in genetic
engineering is reverse transcriptase, an enzyme involved in the
replication of several kinds of virus. Reverse transcriptase is
unique in that it uses as a template not DNA but RNA (Figure
4.5d). The ability of this enzyme to synthesize a DNA strand
complementary to an RNA template is central to the technique
called complementary DNA (cDNA) cloning
54. Figure 4.5 The reactions
catalyzed by DNA
polymerases. (a) The basic
reaction: a new DNA strand is
synthesized in the 5ʹ to 3ʹ
direction. (b) DNA polymerase
I, which initially fills in nicks
but then continues to
synthesize a new strand,
degrading the existing one as
it proceeds. (c) The Klenow
fragment, which only fills in
nicks. (d) Reverse
transcriptase, which uses a
template of RNA.
55. DNA modifying enzymes
There are numerous enzymes that modify DNA molecules by addition
or removal of specific chemical groups. The most important are as
follows:
Polynucleotide phosphorylase
DNAse
Alkaline phosphatase
Polynucleotide kinase
Terminal deoxy nucleotidyl
transferase Methylase
RNAse
56. Polynucleotide phosphorylase
• Polynucleotide Phosphorylase (PNPase) is a bifunctional enzyme
with a phosphorolytic 3' to 5' exoribonuclease activity and a 3'-
terminal oligonucleotide polymerase activity.
• PNPase is a bifunctional enzyme and functions in mRNA processing
and degradation inside the cell.
• Structural and physiochemical studies in enzymes showed that it is
formed of subunits. The arrangements of the subunits may vary
from species to species which would alter their properties.
• These enzyme can catalyze not only the synthesis of RNA from
the mixtures of naturally occurring ribonucleoside diphosphates,
but also that of non-naturally occurring polyribonucleotides
57. Mechanism of action
As mentioned earlier, polynucleotide phosphorylase is a bifunctional enzyme. The
mechanism of action of this enzyme can be represented by following reactions:
In E.coli, polynucleotide phosphorylase regulates mRNA processing
either by
adding ribonucleotides to the 3’ end or by cleaving bases in 3’ to 5’ direction.
The function of PNPase depends upon inorganic phosphate (Pi) concentration inside
the cell.
The transcripts are polyadenylated using enzyme polyadenylate polymerase I (PAPI).
After primary polyadenylylation of the transcript by PAP I, PNPase may bind to the 3ʹ
end of the poly(A) tail. PNPase works either degradatively or biosynthetically inside
the cell depending on the Pi concentration.
Under high Pi concentration, it degrades the poly(A) tail releasing adenine
diphosphates. If the Pi concentration is low, PAP I initiates addition of one or more
nucleotides to the existing poly (A) tail and in the process generates inorganic
phosphate. On dissociation of PNPase, the 3ʹ end again is available to PAP I for further
polymerization.
59. Functions of Polynucleotide phosphorylase
•It is involved in mRNA processing and degradation in bacteria, plants, and in
humans.
•It synthesizes long, highly heteropolymeric tails in vivo as well as accounts for all
of the observed residual polyadenylation in poly(A) polymerase I deficient
strains.
•PNPase function as a part of RNA degradosome in E.coli cell. RNA
degradosome is a multicomponent enzyme complex that includes RNaseE
(endoribinuclease), polynucleotide phosphorylase (3’ to 5’ exonuclease), RhlB
helicase (a DEAD box helicase) and a glycolytic enzyme enolase. This complex
catalyzes 3’ to 5’ exonuclease activity in presence of ATP. Degradsomes in bacteria
are associated with processing, control and turnover of RNA transcripts.
•In rDNA cloning technology, it has been used to synthesize radiolabelled
polyribonucleotides from nucleoside diphosphate monomers.
60. Deoxyribonuclease (DNase):
Deoxyribonuclease (DNase):
A nuclease enzyme that can catalyze the hydrolytic cleavage of phosphodiester bonds in the
DNA backbone are known as deoxyribonuclease (DNase).
Based on the position of action, these enzymes are broadly classified as
endodeoxyribonuclease (cleave DNA sequence internally) and exodeoxyribonuclease (cleave
the terminal nucleotides).
Unlike restriction enzymes, DNase does not have any specific recognition/restriction site and
cleave DNA sequence at random locations.
There is a wide variety of deoxyribonucleases known which have different substrate
specificities, chemical mechanisms, and biological functions. They are:
▪ DNAse I
▪ DNAse II
▪ Exonuclease III
▪ Mung Bean nuclease
61. Deoxyribonuclease I (DNaseI):
An endonuclease which cleaves double-stranded DNA or single stranded DNA.
The cleavage preferentially occurs adjacent to pyrimidine (C or T) residues. The major
products are 5'-phosphorylated bi-, tri- and tetranucleotides.
It requires divalent ions (Ca2+ and Mn2+/Mg2+) for its activity and creates blunt ends or 1-2
overhang sequences.
DNaseI is the most widely used enzyme in cloning experiments to remove DNA
contamination from mRNA preparation (to be used for cDNA library preparation, northern
hybridization, RT-PCR etc). The mode of action of DNaseI varies according to the divalent
cation used.
In the presence of magnesium ions (Mg+2), DNaseI hydrolyzes each strand of duplex DNA
producing single stranded nicks in the DNA backbone, generating various random cleavages.
On the other hand, in the presence of manganese ions (Mn+2), DNaseI cleaves both strands
of a double stranded DNA at approximately the same site, producing blunt ended DNA
fragments or with 1-2 base overhangs.
The two major DNases found in metazoans are: deoxyribonuclease I and deoxyribonuclease II
62. Some of the common applications of DNase I in rDNA technology have been mentioned
below:
• Eliminating DNA contamination (e.g. plasmid) from preparations of RNA.
• Analyzing the DNA-protein interactions via DNA footprinting.
• Nicking DNA prior to radio-labeling by nick translation.
Action of DNase I in the presence of
Mg+2 and Mn+2 ions. (Arrowhead
denoting random site of cleavage in
double stranded DNA by DNase I)
63. DeoxyribonucleaseII (DNaseII)
It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5) and conserved from
human to C.elegans.
It does not require any divalent cation for its activity.
DNaseII initially introduces multiple single stranded nicks in DNA backbone and finally generates 3’
phosphate groups by hydrolyzing phosphodiester linkages.
This enzyme releases 3’phosphate groups by hydrolyzing phosphodiester linkage and creating nicks in
the DNA backbone.
DNaseII acts by generating multiple single stranded nicks followed by production of acid soluble
nucleotides and oligonucleotides.
The catalytic site of the enzyme contains three histidine residues which are essential for
enzyme activity.
Some of the common applications of DNase II are as follows:
• DNA fragmentation
• Molecular weight marker
• Cell apoptosis assays etc.
64. Exonuclease III
Exonuclease III is a globular enzyme which has 3’→5’ exonuclease activity in
a double stranded DNA.
The template DNA should be double stranded and the enzyme does not
cleave single stranded DNA. The enzyme shows optimal activity with blunt ended
sequences or sequences with 5’ overhang.
Exonuclease III enzyme has a bound divalent cation which is essential for enzyme
activity. The mechanism of the enzyme can be affected by variation in temperature,
monovalent ion concentration in the reaction buffer, and structure and
concentration of 3’termini. The enzyme shows optimal activity at 37°C at pH 8.0.
Various application of exonuclease III in molecular cloning experiments are:
• To generate template for DNA sequencing
• To generate substrate for DNA labeling experiments
• Directed mutagenesis
•DNA-protein interaction assays (to find blockage of exonuclease III activity by
protein-DNA binding) etc.
65. Mung bean nuclease
As the name suggest, this nuclease enzyme is isolated from mung bean sprouts (Vigna radiata).
Mung bean nuclease enzymes can degrade single stranded DNA as well RNA.
Under high enzyme concentration, they can degrade double stranded DNA, RNA or
even DNA/RNA hybrids.
Mung bean nuclease can cleave single stranded DNA or RNA to produce 5’-phosphoryl mono
and oligonucleotides.
It requires Zn2+ ion for its activity and shows optimal activity at 37°C.
The enzyme works in low salt concentration (25mM ammonium acetate) and acidic pH (pH
5.0).
Treatment with EDTA or SDS results in irreversible inactivation of the
enzyme. Mung bean nuclease is less robust than S1 nuclease and easier to
handle.
It has been used to create blunt end DNA by cleaving protruding ends from 5’ ends. This
66. Phosphatase
Phosphatase catalyses the cleavage of a phosphate (PO4-2
) group from substrate
by using a water molecule (hydrolytic cleavage).
This reaction is not reversible.
On the basis of their activity there are two types of phosphatase i.e acid
phosphatase and alkaline phosphatase.
In both forms the alkaline phosphatase are most common.
Special class of phosphatase that remove a phosphate group from protein, called
“Phosphoprotein phosphatase”.
Acid phosphatase:
It shows its optimal activity at pH between 3 and 6, e.g. a lysosomal enzyme that
hydrolyze organic phosphates liberating one or more phosphate groups. They are
found in prostatic epithelial cells, erythrocyte, prostatic tissue, spleen, kidney etc
67. Alkaline
phosphatase
Homodimeric enzyme which catalyzes reactions like hydrolysis and trans
phosphophorylation of phosphate monoester.
They show their optimal activity at pH of about 10.
Alkaline phosphatase was the first zinc enzyme discovered having three closed
spaced metal ion. Two Zn+2 ions and one Mg+2 ion, in which Zn+2 ions are bridged
by Asp 51.
In human body it is present in four isoforms, in which three are tissue specific
isoform i.e. placental, germ cell, intestinal and one is non tissue specific isoform.
During post-translational modification, alkaline phosphatase is modified by N-
glycosylation. It undergoes a modification through which uptake of two Zn+2 ion
and one Mg+2 ion occurs which is important in forming active site of that enzyme.
68. Types of Alkaline phosphatase
There are several AP that are used in gene manipulation-
Bacterial alkaline phosphatase (BAP) - Bacterial alkaline phosphatase is a phospho
monoester that hydrolyzes 3’ and 5’ phosphate from nucleic acid (DNA/ RNA).
It more suitably removes phosphate group before end labeling and remove
phosphate from vector prior to insert ligation.
BAP generally shows optimum activity at temperature 65°C.
BAP is sensitive to inorganic phosphate so in presence of inorganic phosphates
activity may reduce.
Calf intestinal alkaline phosphatase (CIP) – It is isolated from calf intestine,
which catalyzes the removal of phosphate group from 5’ end of DNA as well as
RNA.
This enzyme is highly used in gene cloning experiments, as to make a
construct that could not undergo self-ligation.
Hence after the treatment with CIP, without having a phosphate group at 5’ ends a
vector cannot self ligate and recircularise. This step improves the efficiency of vector
containing desired insert
69. Shrimp alkaline phosphatase (SAP) - Shrimp alkaline phosphatase is
highly specific, heat labile phosphatase enzyme isolated from arctic
shrimp (Pandalus borealis). It removes 5’ phosphate group from
DNA, RNA, dNTPs and proteins.
SAP has similar specificity as CIP but unlike CIP, it can be
irreversibly inactivated by heat treatment at 65°C for 15mins.
SAP is used for 5’ dephosphorylation during cloning experiments
for various application as follows:
Dephosphorylate 5’-phosphate group of DNA/RNA for subsequent
labeling of the ends.
To prevent self-ligation of the linearized plasmid.
To prepare PCR product for sequencing.
To inactivate remaining dNTPs from PCR product (for
downstream sequencing appication).
70. Methylase
Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to
its substrate. The process of transfer of methyl group to its substrate is called
methylation
Methylation is a common phenomenon in DNA and protein structure.
Methyltransferase uses a reactive methyl group that is bound to sulfur in Sadenosyl
methionine (SAM) which acts as the methyl donor.
Methylation normally occurs on cytosine (C) residue in DNA sequence.
In protein, methylation occurs on nitrogen atom either on N-terminus or on the
side chain of protein.
DNA methylation regulates gene or silence gene without changing DNA sequences,
as a part of epigenetic regulation.
In bacterial system, methylation plays a major role in preventing their genome from
degradation by restriction enzymes. It is a part of restriction – modification system
in bacteria.
71. Polynucleotide Kinase
PNK has the reverse effect to alkaline phosphatase, adding phosphate groups onto
free 5ʹ termini.
The basic residues of active site of PNK interact with the negatively charged
phosphates of the DNA.
Polynucleotide kinase (PNK) catalyzes the transfer of a phosphate group (PO4-2)
from γ position of ATP to the 5' end of either DNA or RNA and nucleoside
monophosphate.
PNK can convert 3' PO4/5' OH ends into 3' PO4/5' PO4 ends which blocks further
ligation by ligase enzyme.
PNK is used to label the ends of DNA or RNA with radioactive phosphate group.
T4 polynucleotide kinase is the most widely used PNK in molecular cloning
experiments, which was isolated from T4 bacteriophage infected E.coli.
72. PNK carries out two types of enzymatic activity:
•Forward reaction: γ-phosphate is transferred from ATP to the 5' end of a
polynucleotide (DNA or RNA). 5’ phosphate is not present either due to chemical
synthesis or dephosphorylation.
•Exchange reaction: target DNA or RNA having a 5' phosphate is incubated with an
excess of ADP - where PNK transfers the phosphate from the nucleic acid to an ADP,
forming ATP. PNK then performs a forward reaction and transfer a phosphate from ATP to
the target nucleic acid. Exchange reaction is used to label with radioactive phosphate
group. The efficiency of phosphorylation is less in exchange reaction compared to
forward reaction. Along with the phosphorylating activity, PNK also has 3' phosphatase
activity.
73. Uses of PNK
•The linkers and adopters are phosphorylated along
with the fragments of DNA before ligation, which
requires a 5' phosphate. This includes products of
polymerase chain reaction, which are generated by
using non-phosphorylated primers.
•PNK is also used for radio labelling oligonucleotides,
generally with 32P for preparing hybridization probes.
74. Ribonuclease (RNase)
Nuclease that can catalyze hydrolysis of ribonucleotides
from either single stranded or double stranded RNA
sequence are called ribonucleotides (RNase).
•RNase are classified into two types depending on position of
cleavage, i.e.endoribonuclease (cleave internal bond) and
exoribonuclease (cleave terminal bond).
• RNase is important for RNA maturation and processing.
•RNaseA and RNaseH play important role in initial defence
mechanism against RNA viral infection.
75. Types of RNAse
RibonucleaseA (RNaseA):
An endo-ribonuclease that cleaves specifically single-stranded RNA at the 3' end
of pyrimidine residues.
The RNA is degraded into 3'-phosphorylated mononucleotides C and U residues and
oligonucleotides in the form of 2', 3'-cyclic monophosphate intermediates.
Optimal temperature for RNaseA is 60˚C (activity range 15-70˚C) and optimal pH is 7.6
It is used to remove RNA contamination from DNA sample
RibonucleaseH:
Non-specific endoribonuclease that degrades RNA by hydrolytic mechanism from DNA/RNA
duplex resulting in single stranded DNA.
Enzyme bound divalent metal ion is a cofactor here. The product formed is 5’
phosphorylated ssDNA.
During cDNA library preparation from RNA sample, RNaseH enzyme is used to cleave RNA
strand of DNA-RNA duplex.