Genomic and cDNA libraries provide a collection of DNA fragments that can be screened to find genes of interest. To make a genomic library, genomic DNA is purified from cells, fragmented, and cloned into a vector such as lambda phage. A large number of clones are needed to represent the entire genome. cDNA libraries are made from mRNA which is isolated, reverse transcribed to cDNA, and cloned into a vector. Libraries are screened by hybridization, expression analysis or other methods to identify clones containing genes of interest. Repeated screening allows chromosome walking to obtain overlapping genomic fragments.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Chromosome walking is a method used to locate and clone a specific gene or allele through successive identification of overlapping DNA sequences. It begins at a known marker gene near the target and "walks" through testing genes one by one to map their locations and identify overlaps, eventually reaching the mutant gene. Once the full sequence is cloned, the gene's function can be determined to study genetically transmitted diseases. It is a complex process but has allowed mapping of large chromosome regions over 1000kb.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
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.
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Lectut btn-202-ppt-l20. genomic and c dna librariesRishabh Jain
Genomic and cDNA libraries are collections of clones containing DNA fragments from an organism. A genomic DNA library contains all fragments of the genomic DNA, while a cDNA library contains only coding sequences synthesized from expressed mRNA. Genomic libraries are suitable for prokaryotes due to their small genomes but eukaryotic genomes require too many clones, so cDNA libraries are preferred for eukaryotic gene cloning. cDNA libraries represent the expressed genes and contain only coding regions without introns.
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Chromosome walking is a method used to locate and clone a specific gene or allele through successive identification of overlapping DNA sequences. It begins at a known marker gene near the target and "walks" through testing genes one by one to map their locations and identify overlaps, eventually reaching the mutant gene. Once the full sequence is cloned, the gene's function can be determined to study genetically transmitted diseases. It is a complex process but has allowed mapping of large chromosome regions over 1000kb.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
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.
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Lectut btn-202-ppt-l20. genomic and c dna librariesRishabh Jain
Genomic and cDNA libraries are collections of clones containing DNA fragments from an organism. A genomic DNA library contains all fragments of the genomic DNA, while a cDNA library contains only coding sequences synthesized from expressed mRNA. Genomic libraries are suitable for prokaryotes due to their small genomes but eukaryotic genomes require too many clones, so cDNA libraries are preferred for eukaryotic gene cloning. cDNA libraries represent the expressed genes and contain only coding regions without introns.
The document discusses the process of synthesizing cDNA from mRNA. It involves isolating mRNA, using reverse transcriptase to copy the mRNA into single-stranded cDNA, then converting it to double-stranded cDNA using DNA polymerase. The double-stranded cDNA can then be inserted into a vector and used to create a cDNA library through cloning in bacteria or phage. The library can be screened by hybridization or assays to identify clones containing genes of interest.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
PHYSICAL MAPPING STRATEGIES IN GENOMICSUsman Arshad
Genetic and physical mapping are two types of genome mapping. Genetic mapping uses pedigree analysis and breeding experiments to determine sequence features, while physical mapping uses molecular techniques. Restriction mapping, radiation hybrid mapping, and STS mapping are techniques used to construct physical maps in the absence of complete DNA sequencing. Restriction mapping identifies restriction sites, radiation hybrid mapping analyzes fragments from irradiated cells hybridized with hamster cells, and STS mapping tags genomic sites using PCR primers. These physical mapping strategies provide distance and order estimates between DNA sequences to construct frameworks for sequencing.
The document discusses various methods for synthesizing complementary DNA (cDNA) from messenger RNA (mRNA). It describes the basic three step process of first-strand cDNA synthesis using reverse transcriptase, removal of the RNA template, and second-strand cDNA synthesis using DNA polymerase. Early methods used hairpin priming of the second strand but were later improved using oligo-dT tailing and oligo-dG priming to avoid 5' end losses. Other methods discussed include oligo-capping to select for full-length mRNAs and RACE (rapid amplification of cDNA ends) to amplify cDNA fragments from both ends of transcripts.
Cloning and Expression Vectors document discusses:
1) Cloning a gene of interest involves inserting it into a vector that can be replicated in host cells, producing recombinant DNA molecules.
2) Vectors contain features like replication origins, antibiotic resistance genes, and unique restriction sites to facilitate cloning and isolation.
3) Early cloning experiments demonstrated that recombinant plasmids containing both prokaryotic and eukaryotic DNA could replicate stably in bacteria, allowing genetic engineering.
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.
Whole genome shotgun sequencing involves randomly breaking genomic DNA into small fragments, sequencing the fragments, and then reassembling the sequences using overlapping regions. The document outlines the history and procedure of shotgun sequencing. Genomic DNA is first fragmented, end-repaired, and size-selected into small, medium, and large fragments. Libraries are created for each size fragment and sequenced. A base caller filters poor calls and an assembler finds overlaps to generate continuous nucleotide sequences or contigs of the whole genome.
Structural genomics is a field that aims to determine the 3D structures of all proteins encoded by a genome. It involves determining structures on a large scale using techniques like X-ray crystallography and NMR. This allows identification of novel protein folds and potential drug targets. Comparative genomics compares genomic features between organisms and provides insights into evolution and conserved sequences and functions. It is a key tool in fields like medicine and agriculture.
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.
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
This document discusses cDNA and genomic libraries. It defines cDNA as complementary DNA synthesized from mRNA, and genomic libraries as collections of DNA fragments representing an organism's entire genome. It describes how cDNA libraries are constructed by synthesizing cDNA from purified mRNA, and genomic libraries by partially digesting genomic DNA and inserting fragments into cloning vectors. It also explains how these libraries are screened using probes to identify clones containing genes of interest or encoding specific proteins.
CDNA Library preparation. ppt for Jamil sirNushrat Jahan
cDNA is produced from mRNA found in the nucleus and contains only the expressed genes of an organism. To create a cDNA library, mRNA is first extracted and purified from a cell, then reverse transcribed into cDNA using an oligo-dT primer that binds to the poly-A tail. The resulting single-stranded cDNA is converted into double-stranded DNA and cloned into plasmids in bacteria. cDNA libraries are useful for reproducing eukaryotic genomes without introns, expressing eukaryotic genes in prokaryotes, discovering novel genes, and studying alternative splicing in different cells.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
PHYSICAL MAPPING STRATEGIES IN GENOMICSUsman Arshad
Genetic and physical mapping are two types of genome mapping. Genetic mapping uses pedigree analysis and breeding experiments to determine sequence features, while physical mapping uses molecular techniques. Restriction mapping, radiation hybrid mapping, and STS mapping are techniques used to construct physical maps in the absence of complete DNA sequencing. Restriction mapping identifies restriction sites, radiation hybrid mapping analyzes fragments from irradiated cells hybridized with hamster cells, and STS mapping tags genomic sites using PCR primers. These physical mapping strategies provide distance and order estimates between DNA sequences to construct frameworks for sequencing.
The document discusses various methods for synthesizing complementary DNA (cDNA) from messenger RNA (mRNA). It describes the basic three step process of first-strand cDNA synthesis using reverse transcriptase, removal of the RNA template, and second-strand cDNA synthesis using DNA polymerase. Early methods used hairpin priming of the second strand but were later improved using oligo-dT tailing and oligo-dG priming to avoid 5' end losses. Other methods discussed include oligo-capping to select for full-length mRNAs and RACE (rapid amplification of cDNA ends) to amplify cDNA fragments from both ends of transcripts.
Cloning and Expression Vectors document discusses:
1) Cloning a gene of interest involves inserting it into a vector that can be replicated in host cells, producing recombinant DNA molecules.
2) Vectors contain features like replication origins, antibiotic resistance genes, and unique restriction sites to facilitate cloning and isolation.
3) Early cloning experiments demonstrated that recombinant plasmids containing both prokaryotic and eukaryotic DNA could replicate stably in bacteria, allowing genetic engineering.
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.
Whole genome shotgun sequencing involves randomly breaking genomic DNA into small fragments, sequencing the fragments, and then reassembling the sequences using overlapping regions. The document outlines the history and procedure of shotgun sequencing. Genomic DNA is first fragmented, end-repaired, and size-selected into small, medium, and large fragments. Libraries are created for each size fragment and sequenced. A base caller filters poor calls and an assembler finds overlaps to generate continuous nucleotide sequences or contigs of the whole genome.
Structural genomics is a field that aims to determine the 3D structures of all proteins encoded by a genome. It involves determining structures on a large scale using techniques like X-ray crystallography and NMR. This allows identification of novel protein folds and potential drug targets. Comparative genomics compares genomic features between organisms and provides insights into evolution and conserved sequences and functions. It is a key tool in fields like medicine and agriculture.
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.
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
This document discusses cDNA and genomic libraries. It defines cDNA as complementary DNA synthesized from mRNA, and genomic libraries as collections of DNA fragments representing an organism's entire genome. It describes how cDNA libraries are constructed by synthesizing cDNA from purified mRNA, and genomic libraries by partially digesting genomic DNA and inserting fragments into cloning vectors. It also explains how these libraries are screened using probes to identify clones containing genes of interest or encoding specific proteins.
CDNA Library preparation. ppt for Jamil sirNushrat Jahan
cDNA is produced from mRNA found in the nucleus and contains only the expressed genes of an organism. To create a cDNA library, mRNA is first extracted and purified from a cell, then reverse transcribed into cDNA using an oligo-dT primer that binds to the poly-A tail. The resulting single-stranded cDNA is converted into double-stranded DNA and cloned into plasmids in bacteria. cDNA libraries are useful for reproducing eukaryotic genomes without introns, expressing eukaryotic genes in prokaryotes, discovering novel genes, and studying alternative splicing in different cells.
Dna library lecture-Gene libraries and screening Abdullah Abobakr
This document discusses gene libraries and screening procedures. It begins by explaining what genomic and cDNA libraries are. It then provides details on creating genomic libraries, including purifying genomic DNA, fragmenting it, and cloning the fragments into vectors. Creating cDNA libraries involves isolating mRNA, synthesizing cDNA, and ligating the cDNA to vectors. The size of libraries needed to ensure coverage of genomes is calculated. Lambda phage is described as a commonly used vector that can accept inserts up to 23kb in size. The processes of packaging recombinant DNA into lambda phage particles and creating lambda phage libraries are outlined.
The three sentence summary is:
The Human Genome Project aimed to map and sequence all the genes in the human genome between 1990-2003 through international collaboration, with the goals of identifying all human genes, determining their sequences and mapping genetic variations; two main strategies were used including a public hierarchical shotgun approach and a private whole genome shotgun by Celera; the results provided the complete human genome sequence and understanding of human genetic content and variation with applications to medicine like disease gene identification and new drug targets.
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.
Applications of genetic engineering techniques in agricultureB.Devadatha datha
This document discusses applications of genetic engineering techniques in agriculture. It begins by outlining reasons for genetically engineering plants, such as improving crops, using plants as bioreactors, and studying gene action. Various genetic engineering methods are then described, including Agrobacterium-mediated gene transfer. Applications like developing herbicide-resistant, insect-resistant, virus-resistant, and drought/cold-tolerant crops are covered. The document also discusses using genetic engineering for nutritional enhancement and production of edible vaccines. Potential risks like contamination and effects on non-target organisms are noted.
DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.
The document discusses the human genome project, which aimed to sequence the entire human genome and identify all human genes. It provides background on the human genome, describing its size, number of genes, and chromosomes. It details the goals and milestones of the human genome project from 1986 to 2003. Vectors like yeast artificial chromosomes and bacterial artificial chromosomes were used to clone large fragments of DNA for sequencing.
This document summarizes different methods for screening DNA libraries to identify specific clones. It discusses screening by hybridization using radioactive probes or alternative labeling methods. It also describes screening by PCR using gene-specific primers and screening expression libraries using antibodies that recognize antigenic determinants on expressed polypeptides. Screening methods like Southwestern and Northwestern blotting combine principles of Southern/Western blots to identify DNA or RNA binding proteins. The goal of these screening methods is to efficiently identify clones containing specific DNA sequences or expressing desired proteins from large DNA libraries.
The document provides information on principles of cell culture. It discusses the history of cell culture, beginning with Roux maintaining embryonic chick cells in 1885. It describes the typical equipment used in cell culture like laminar flow cabinets and incubators. The document outlines the different types of cell cultures like primary cultures derived directly from tissue and continuous cultures that can be serially passaged. It explains concepts like passaging cells to maintain and expand cultures. The document also discusses cryopreservation to store cells long-term in liquid nitrogen. Common cell lines used in research are also mentioned like HeLa and MCF-7 cells. Contamination in cell culture can occur from bacteria, fungi or mycoplasma and affect cell growth. Strict aseptic
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
Genomic DNA and cDNA libraries can be generated to study the entire genome or expressed genes. Genomic libraries contain all DNA including exons and introns, while cDNA libraries contain only expressed exons. λ-phage is commonly used as the vector since it can package large DNA fragments and be screened efficiently. The process involves fragmenting DNA, ligating it into λ-phage arms, packaging it into phage particles which are then plated and screened to isolate clones across the genome. cDNA is generated from mRNA and also ligated into λ-phage to create a library representing the expressed genes.
Genomic DNA and cDNA libraries can be generated using lambda phage (λ-phage) as a vector. λ-phage is useful because it can package large DNA fragments, has a high transformation efficiency, and replicates lysogenically. To generate a genomic library, genomic DNA is fragmented, ligated into λ-phage arms, and packaged into phage particles. A cDNA library contains only expressed sequences; it is made by isolating mRNA, reverse transcribing it to cDNA, and ligating the cDNA into λ-phage arms. Both library types allow cloning and sequencing of large genomic regions.
The document discusses gene cloning, expression, and functional study. It describes different types of vectors used for cloning genes, including cloning vectors, expression vectors, and integration vectors. It provides details on various cloning vectors such as plasmid vectors, bacteriophage vectors, cosmids, BACs, and eukaryotic vectors. It also describes expression vectors and components required for gene expression. Finally, it discusses bacteriophage vectors, cosmids, YAC vectors, and BAC vectors which are used to clone large DNA fragments from eukaryotes.
Genomic DNA and cDNA libraries are two ways to obtain genomic information. [1] A genomic DNA library contains the entire human genome, including exons and introns. [2] A cDNA library contains only the expressed portions of the genome (exons). [3] λ-phage is commonly used as the vector to generate these libraries due to its ability to clone large DNA fragments and be packaged in large numbers.
This document discusses DNA libraries, which are collections of cloned DNA sequences from an organism. There are two main types: genomic libraries, made from genomic DNA, and cDNA libraries, made from complementary DNA (cDNA) synthesized from mRNA. Genomic libraries contain whole genes including introns and regulatory elements, while cDNA libraries contain only mRNA transcripts without introns or untranslated regions. Common vectors used for cloning DNA include plasmids, bacteriophages, and yeast artificial chromosomes. The construction of genomic and cDNA libraries involves isolating DNA or mRNA, fragmenting, cloning fragments into vectors, and inserting the vectors into bacteria. Genomic and cDNA libraries have various applications including gene identification, expression of eukaryotic genes in
Principle and procedure for making Genomic library and cDNA library.pptxPrabhatSingh628463
This document presents information on genomic and cDNA libraries. It begins by defining a gene library as a collection of DNA sequences from an organism cloned into a vector. There are two main types - genomic libraries containing all sequences from the genome, and cDNA libraries containing sequences represented in mRNA. The document then describes the principles, vectors, and procedures used to construct each type of library. Key steps include fragmenting genomic DNA, ligating fragments into vectors, and amplifying the libraries in host cells for genomic libraries, and reverse transcribing mRNA and ligating cDNA into vectors for cDNA libraries. The advantages and disadvantages of each approach are also summarized.
Construction of genomic and c dna libraryNaveenJ46
The document summarizes the process of constructing genomic and cDNA libraries. A genomic library contains total genomic DNA from an organism, including exons and introns. Genomic DNA is extracted, fragmented, and inserted into vectors which are then introduced into host cells. Each cell contains a different DNA fragment. A cDNA library contains only expressed genes as cDNA, excluding introns. mRNA is extracted and reverse transcribed to cDNA, which is then inserted into vectors. Both library types are useful tools for sequencing and studying gene expression.
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
This document discusses cDNA and genomic libraries. It defines cDNA and genomic libraries and explains their key differences. A cDNA library contains only complementary DNA molecules synthesized from mRNA in a cell and represents the genes expressed in that cell. Genomic libraries for prokaryotes are easier to make and contain all genome sequences since prokaryotic mRNA is unstable. cDNA libraries are useful for eukaryotic gene analysis as they contain condensed protein-encoded genes without introns. The document also provides details on the construction, screening, and uses of cDNA and genomic libraries.
Genomic and cDNA libraries are collections of DNA fragments used for gene discovery and analysis. cDNA libraries contain only expressed genes and are useful for eukaryotic analysis since they lack introns. Genomic libraries contain all DNA sequences from an organism's genome. Both types of libraries are constructed by fragmenting DNA, inserting fragments into cloning vectors, and transforming bacteria to generate clones containing DNA fragments. Libraries are screened using probes to identify clones containing genes of interest.
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
This document discusses genomic and cDNA libraries. Genomic libraries are made from genomic DNA and represent all genes in an organism. They require a minimum number of clones to ensure all genes are captured. cDNA libraries are made from mRNA and represent expressed genes, avoiding introns. Key steps in making cDNA libraries include mRNA isolation, cDNA synthesis, addition of linkers, and ligation into a vector. Screening methods to identify clones of interest include hybridization, expression screening, and hybrid arrest/release.
This document discusses genomic DNA libraries and cDNA libraries. It provides information on:
- Genomic DNA libraries contain DNA fragments representing an organism's entire genome, created using molecular cloning. cDNA libraries contain DNA copies of expressed genes from mRNA.
- The process of constructing a genomic library involves isolating genomic DNA, cutting it into fragments, inserting the fragments into vectors, and adding them to bacteria to form a library.
- cDNA libraries are constructed from mRNA. The process involves isolating mRNA, converting it to cDNA using reverse transcriptase, inserting the cDNA into vectors, and cloning the vectors into host cells.
Genomic and cDNA libraries allow for the representation of genomic sequences as multiple small fragments. A genomic library contains fragments from all DNA sources, including coding and non-coding regions. A cDNA library contains only expressed coding sequences, as it is synthesized from mRNA. The process involves isolating mRNA, synthesizing cDNA, incorporating it into a vector, and cloning the fragments. Libraries are important tools for studying genomes, genes, and gene expression.
This document describes DNA libraries and the process of creating them. It discusses genomic libraries, which contain an organism's entire genome, and cDNA libraries, which contain copies of mRNA. The key steps to create a library are: 1) isolating and fragmenting DNA, 2) cloning the fragments into vectors, 3) transforming the vectors into host cells, 4) multiplying and screening the clones to identify fragments of interest. cDNA libraries are useful for studying eukaryotic genes as they remove non-coding regions.
Genomic DNA libraries contain representative copies of all DNA fragments in an organism's genome, including both expressed and non-expressed sequences. They are constructed by isolating genomic DNA, fragmenting it, and cloning the fragments into suitable vectors like lambda phage or BACs. cDNA libraries contain only expressed sequences, as they are constructed by isolating mRNA from tissues, reverse transcribing it to cDNA, and cloning the cDNA fragments. Both library types are useful for gene discovery, sequencing, mapping genomes, and studying regulatory sequences.
Gene libraries, such as cDNA and genomic libraries, allow isolation of specific genes. cDNA libraries contain only exons and reflect gene expression levels, while genomic libraries contain all DNA fragments. Libraries are constructed by fragmenting DNA and cloning into vectors before transforming bacteria. They can be screened by hybridization, PCR, or immunological assays to detect gene products. Common steps include lysis, fixation, and detection to identify positive clones containing genes of interest.
Gene libraries play a central role in gene cloning experiments by providing a collection of clones representing an organism's entire genome. There are two main types of gene libraries - genomic libraries and cDNA libraries. Genomic libraries are constructed by fragmenting genomic DNA and cloning the fragments into vectors. cDNA libraries contain DNA copies of mRNA produced from reverse transcribing isolated mRNA. Both library types allow screening to identify specific genes of interest from the organism. The size and probability of a library containing the desired gene determines the number of clones needed in the library.
DNA libraries allow for the storage and organization of genetic information, similar to how physical libraries store books. There are two main types of DNA libraries: genomic libraries, which are created from genomic DNA and contain entire genes with exons and introns, and cDNA libraries, which are created from mRNA and contain only exons. To create a genomic library, genomic DNA is isolated, fragmented, and inserted into cloning vectors within host bacteria. For cDNA libraries, mRNA is isolated, reverse transcribed into cDNA, which is then amplified and inserted into vectors. Both library types are screened to find clones containing desired DNA sequences.
1. Molecular cloning involves cutting DNA from one organism and inserting it into a vector that can replicate in a host organism, allowing the DNA fragment to be amplified. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments that are then ligated into cloning vectors like plasmids or bacteriophages.
2. After transforming host bacteria with the recombinant vector, clones containing the inserted DNA fragment can be selected for and amplified. Colonies containing the insert are identified through antibiotic resistance or colorimetric markers present on the vector.
3. cDNA libraries provide a way to clone and study eukaryotic genes. mRNA is isolated and reverse transcribed into cDNA, which is then ligated into vectors and
This document discusses gene regulation in prokaryotes and eukaryotes. It explains that gene regulation allows cells to only express genes when they are needed. In prokaryotes, gene regulation typically occurs through operons at the transcriptional level. Eukaryotic gene regulation is more complex and can occur through epigenetic, transcriptional, post-transcriptional, translational and post-translational mechanisms. Key methods of regulation include chromatin remodeling, transcription factor binding, RNA processing, mRNA degradation, and protein degradation.
Ribosomes are sub-microscopic organelles found in all living cells that are the sites of protein synthesis. They are composed of ribosomal RNA and proteins. Ribosomes exist in two types - 70S ribosomes in prokaryotes and 80S ribosomes in eukaryotes. 70S ribosomes are composed of a 50S and 30S subunit while 80S ribosomes have a 60S and 40S subunit. Ribosomes contain three functional sites - the mRNA binding site, the aminoacyl-tRNA site (A site), and the peptidyl-tRNA site (P site) where protein synthesis occurs through the joining of amino acids. Ribosomes provide enzymes and factors to facilitate the
Vacuoles are membrane-bound organelles found in plant and fungal cells that function to store waste, nutrients, and water to regulate pressure within the cell. The cell wall is located outside the plasma membrane in plant, fungal, and some bacterial and algal cells and provides structure, protection, and allows for cell growth and communication. The nucleus houses the cell's genetic material and has a surrounding membrane with pores to allow transport of molecules in and out. Peroxisomes are spherical organelles found in all eukaryotic cells that contain enzymes for metabolic processes. The cytosol is the liquid within the cell and cytoplasm but outside organelles, and serves functions of transport, metabolism, and signaling.
Biotechnology has been used for millennia to improve agriculture, food production, and medicine through techniques like animal husbandry and fermentation. Modern biotechnology applies scientific principles to processing materials through biological agents. It has applications in medicine like drug development, agriculture like developing pest-resistant crops, and industry like producing chemicals. Biotechnology's scope continues expanding in fields such as genetic engineering, stem cell research, and environmental remediation.
unit 1 cytoskeletal structures ECM docx.pdf sh.pdfMSCW Mysore
The cytoskeleton is made up of three main components - microtubules, microfilaments, and intermediate filaments. Microtubules are hollow tubes made of tubulin dimers that help maintain cell shape and transport vesicles. Microfilaments are made of actin and enable cell movement and division. Intermediate filaments provide structural support and anchor organelles. The extracellular matrix of animal cells contains collagen, fibronectin and proteoglycans, while plant cells have a cell wall made of cellulose and pectin.
Biotechnology III sem Practical manual MSCW Mysore
This document contains laboratory protocols for experiments in cell biology and genetics. It includes procedures for observing mitosis in onion root tip cells using a squash technique, studying meiosis in onion flower bud cells using a permanent slide, examining Barr bodies in human buccal smear cells, and isolating chloroplasts from spinach leaves and mitochondria from yeast cells through differential centrifugation. The document provides detailed methodologies, materials required, and expected observations for each experiment to analyze key cellular processes like the cell cycle, meiosis, and intracellular organelle isolation.
This document discusses vitamins, classifying them as either fat-soluble or water-soluble. Fat-soluble vitamins include A, D, E, and K and are absorbed with fats. Water-soluble vitamins include vitamin C and the B complex vitamins. The B complex vitamins act as coenzymes in energy production and metabolism. The document provides details on functions, deficiency and toxicity symptoms, and food sources for several important vitamins.
Waste water treatment technology SH/pdfMSCW Mysore
1. The document discusses various strategies for wastewater treatment, including preliminary, primary, secondary, and tertiary treatments.
2. Preliminary treatment involves removing floating materials, settleable solids, and oils/greases using screens, grit chambers, and skimming tanks.
3. Primary treatment uses sedimentation to remove suspended solids via settling, and may use chemicals to aid the process.
The document discusses different types of biomolecules, focusing on carbohydrates. It defines carbohydrates and describes their classification into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The key monosaccharides discussed are glucose, fructose, and galactose. Major disaccharides include sucrose, maltose, and lactose. Carbohydrates can exist in both chain and ring forms.
This document provides information about career options in biotechnology. It discusses the various academic and industry career paths available with undergraduate, postgraduate, and PhD degrees in biotechnology and related fields. Specific job roles are outlined for research assistants, research associates, senior research associates, scientists, principal scientists, scientific directors, and bioinformatics scientists. The document also provides details on the Indian biotechnology industry and job market size, key organizations, and tips for pursuing opportunities in biotechnology.
This document provides a lab manual covering various immunology and cell culture techniques. It includes 12 experiments on topics such as blood grouping, Rh factor testing, hemoglobin estimation, differential leukocyte counting, red blood cell counting using a Neubauer chamber, and total leukocyte counting with a Neubauer chamber. For each experiment, the document discusses the principle, requirements, procedure, results and interpretation. The experiments aim to teach fundamental immunology and cell culture methods relevant to research and clinical applications.
Cell membrane permeability and functionsMSCW Mysore
The document summarizes key aspects of cell membrane structure and function. It describes the fluid mosaic model of the cell membrane, which depicts the membrane as a bilayer of phospholipids embedded with proteins, carbohydrates, cholesterol, and other components. The membrane acts as a semi-permeable barrier, allowing selective transport of molecules into and out of the cell through integral membrane proteins. Specific glycoproteins on the cell surface are exploited by viruses to infect particular cell types and organs. Transport across the cell membrane is mediated by membrane-spanning transport proteins that facilitate movement of ions and molecules.
This document provides lecture notes on DNA replication, damage, and repair. It discusses the process of DNA replication including the three main steps of initiation, elongation, and termination. Details are given on replication in prokaryotes and eukaryotes. The document also covers types of DNA damage including physical, chemical, and biological mutagens. Finally, various DNA repair mechanisms are summarized such as photoreactivation, excision repair, and SOS repair.
Role of genetically engineered microorganisms in biodegradationMSCW Mysore
1. The document discusses biological recovery from oil spills, including the environmental effects of oil spills and various cleanup methods. It also examines the role of genetically engineered microorganisms (GEMs) in biodegrading pollutants.
2. Key cleanup methods include booms, skimming, solidifiers, dispersants, and bioremediation using oil-consuming bacteria. GEMs have been developed to enhance biodegradation by modifying enzyme specificity and constructing new metabolic pathways.
3. While GEMs show potential for degrading various hydrocarbons, field trials of GEMs for bioremediation remain limited due to regulations and public concerns.
structural biology-Protein structure function relationshipMSCW Mysore
Structural biology determines protein structures using x-ray crystallography. X-rays are diffracted by regular arrays of atoms in protein crystals to produce patterns that reveal atomic structures. Protein structures determine their functions, such as catalytic activity. Understanding protein structures is essential to elucidating their roles in cellular processes.
practical manual on molecular biology and genetic engineering,recombinant DNA...MSCW Mysore
This document is a laboratory manual containing protocols for isolating DNA from various eukaryotic sources like onion, liver, coconut, and spleen. It describes the common principle of isolating DNA from nucleoproteins using low salt concentrations, then releasing the DNA from proteins using high salt concentrations, followed by ethanol precipitation to collect the DNA. The manual contains 13 experiments covering DNA and RNA isolation, agarose gel electrophoresis, restriction digestion, and ligation. It is intended as a guide for third year biotechnology students to learn techniques in molecular biology and genetic engineering.
A workshop hosted by the South African Journal of Science aimed at postgraduate students and early career researchers with little or no experience in writing and publishing journal articles.
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
How to Setup Warehouse & Location in Odoo 17 InventoryCeline George
In this slide, we'll explore how to set up warehouses and locations in Odoo 17 Inventory. This will help us manage our stock effectively, track inventory levels, and streamline warehouse operations.
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How to Make a Field Mandatory in Odoo 17Celine George
In Odoo, making a field required can be done through both Python code and XML views. When you set the required attribute to True in Python code, it makes the field required across all views where it's used. Conversely, when you set the required attribute in XML views, it makes the field required only in the context of that particular view.
How to Build a Module in Odoo 17 Using the Scaffold MethodCeline George
Odoo provides an option for creating a module by using a single line command. By using this command the user can make a whole structure of a module. It is very easy for a beginner to make a module. There is no need to make each file manually. This slide will show how to create a module using the scaffold method.
The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
2. Introduction
The use of genetic information is a powerful tool that
today is becoming more readily available to scientists.
In order to use this powerful tool it necessary to know
how to navigate throughout the entire genome. The
human genome is about 3 x 10E9 bp.
In humans this project is known as Human Genome
Project.
3. Gene library: a collection of different DNA
sequence from an organism, each of which
has been cloned into a vector for ease of
purification, storage and analysis.
Genomic libraries
cDNA libraries
Gene library
(made from genomic DNA)
(made from cDNA- copy of mRNA)
I1 Genomic libraries
4. Size of library (ensure enough clones)
must contain a certain number of
recombinants for there to be a high probability
of it containing any particular sequence
The formula to calculate the number of
recombinants:
N =
ln (1-P)
ln (1-f)
P: desired probability
f : the fraction of the genome in one insert
I1 Genomic libraries
5. For example :for a probability of 0.99 with
insert sizes of 20 kb these values for the E.coli
(4.6×106
bp) and human (3×109
bp) genomes
are :
N E.coli= = 1.1 ×103
ln( 1-0.99)
ln[1-(2×104
/4.6×106
)]
Nhuman= = 6.9 ×105
ln(1-0.99)
ln[1-(2 ×104
/3 ×109
)]
These values explain why it is possible to make good
genomic libraries from prokaryotes in plasmids where
the insert size is 5-10kb ,as only a few thousand
recombinants will be needed.
I1 Genomic libraries
6. Genomic DNA libraries
Purify genomic DNA
Fragment this DNA : physical shearing
and restriction enzyme digestion
eukaryotes
prokaryotes
Clone the fragments into vectors
I1 Genomic libraries
7. To make a representative genomic libraries ,
genomic DNA must be purified and then
broken randomly into fragments that are
correct in size for cloning into the chosen vector.
Purification of genomic DNA :
Prokaryotes :extracted DNA directly from cells
remove protein, lipids and other unwanted macro-
molecules by protease digestion and phase extraction.
Eukaryotes :prepare cell nuclei
I1 Genomic libraries
8. Break DNA into fragments randomly:
Physical shearing :
pipeting, mixing
Restriction enzyme digestion:
partial digestion is preferred
to get a greater lengths of DNA
fragments.
I1 Genomic libraries
9. Sau3A: 5’-/GATC-3’, less selectivity
BamH1: 5’-G/GATCC
Selection of restriction enzyme
1. Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
2. Whether the enzyme is inhibited by DNA
modifications (CpG methylation in
mammals
3. Time of digestion and ratio of restriction
enzyme to DNA is dependent on the
desired insert size range.
I1 Genomic libraries
10. Generating A Genomic
Library
λ-phage is treated with restriction
enzymes that produce λ arms with
sticky end. These arms contain all
the lytic genetic information that is
needed for replication and produces
room for insertion of new genetic
information.
DNA sequence is obtain from the cell
of interest. It is cleaved with
restriction enzymes that produce
20kb fragments that have
complementary sticky ends.
Both are mixed in equal amounts and
are treated with a DNA ligase that
cleaves them together.
Afterward the entire combined
sequence is packed to the phage
head.
11. λ-phage as a Vector
The genomic library is
generated by using λ-phage
for the following reasons.
1. A large number of λ phage can be
screened simultaneously (5 x 10E4
phage plagues).
2. λ phage as a higher transformation
efficiency about 1000 times higher
compared to a plasmid.
The vector as to maintain its
lytic growth.
Lysogenic pathway and
other viral genes that are
not important are replaced
with the DNA to be cloned.
12. λ-phage as a Vector (Cont.)
An infected E.Coli will produce
what are know as concatomers
(which is the viral genome) on
either site of the concatomers
there is a site called COS Site.
Two proteins recognize this site
A protein and Nu protein, which
will lead to the insertion of the
λ DNA into the phage head. The
chromosomal DNA that lacks the
COS sites will not enter the
phage head. Once the genetic
information is inserted the tail
will assemble.
A 50kb can be inserted into the
phage.
13. Packaging of the Recombinant DNA
To prepare the phage an E.coli cell is infected with a mutant λ-
phage that as a defective “A-protein” (which is one of two genes
that are responsible for packaging genetic information).
Therefore the E.Coli accumulates empty heads and also
preassembled tails.
Once enough heads and tails are assembled we lysate the E.Coli
cells.
To the mixture of heads and tail we add isolated A protein
(obtained from E.Coli infected with λ-phage).
In the next step we add the recombinant DNA that has the λ-
phage genetic information (which also includes COS sites).
At this point we have a mixture containing mutant λ-phage heads
and tails. There is isolated A protein and recombinant DNA
containing λ-phage genetic information with COS sites.
Therefore we have all the components necessary to package the
recombinant DNA into the λ-phage head. Once the information is
inserted the tail assembles and we have an infectious phage that
contains the recombinant DNA sequence.
14. Vectors
According to genome’s size,we can select a
proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb) 5 23 45 1000
The most commonly chosen genomic cloning vectors
are λ relacement vectors which must be digested with
restriction enzymes to produce the two λ end fragment
or λ arms between which the genomic DNA will be
digested
I1 Genomic libraries
15. cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
λ phage vector in cloning
cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
16. λ replacement
vector cloning
2. Packing with a
mixture of the phage
coat proteins and
phage DNA-
processing enzymes
3. Infection and
formation of
plaques
Library constructed
1. Ligation
0.preparation of
arm and genomic
inserts
17. I cDNA libraries
mRNA isolation, purification
Check theRNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
Gene libraries and screening
18. cDNA libraries
1. No cDNA library was made
from prokaryotic mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes
are easier to make and contain all
the genome sequences.
I 2 cDNA libraries
19. 2. cDNA libraries are very useful
for eukaryotic gene analysis
• Condensed protein encoded gene
libraries, have much less junk sequences.
• cDNAs have no introns → genes can be
expressed in E. coli directly
• Are very useful to identify new genes
• Tissue or cell type specific (differential
expression of genes)
cDNA libraries
I 2 cDNA libraries
20. mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
• oligo (dT) can be bound to the poly(A) tail
and used to recover the mRNA.
AAAAAAAAAAn5’ cap
I 2 cDNA libraries
22. 1.Traditionally method was done by pass a
preparation of total RNA down a column of
oligo (dT)-cellulose
2.More rapid procedure is to add oligo(dT)
linked to magnetic beads directly to a cell
lysate and ‘pulling out’ the mRNA using a
strong magnet
3.Alternative route of isolating mRNA is
lysing cells and then preparing mRNA-
ribosome complexes on sucrose gradients
Three methods to isolate mRNA.
I2 cDNA libraries
23. Make sure that the mRNA is not
degraded. Methods:
Translating the mRNA : use cell-free
translation system as wheat germ extract or
rabbit reticulocyte lysate to see if the mRNAs
can be translated
Analysis the mRNAs by gel
elctrophoresis: use agarose or
polyacrylamide gels
Check the mRNA integrity
I2 cDNA libraries
24. Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to make a complete
cDNA library.
Fractionate on the gel: performed on
the basis of size, mRNAs of the interested
sizes are recovered from agarose gels
Enrichment: carried out by hybridization
Example: clone the hormone induced mRNAs
(substrated cDNA library)
I2 cDNA libraries
25. Synthesis of cDNA :
First stand synthesis: materials as
reverse transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs
Second strand synthesis: best way of
making full-length cDNA is to ‘tail’ the 3’-
end of the first strand and then use a
complementary primer
to make the second.
I2 cDNA libraries
26. 5’ mRNA AAAAA-3’
HO-TTTTTP-5’
5’
Reverse transcriptase
Four dNTPs
AAAAA-3’
TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferase
dCTP
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG
3’-CCCCCCC TTTTTP-5’
-3’
The first strand synthesis
I2 cDNA libraries
28. Treatment of cDNA ends
Blunt and ligation of large fragment is not
efficient, so we have to use special acid linkers to
create sticky ends for cloning.
The process :
Move protruding 3’-ends(strand-special nuclease)
Fill in missing 3’ nucleotide (klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Restriction enzyme digestion (E.coRI )
Tailing with terminal transferase or
using adaptor molecules
I2 cDNA libraries
29. Ligation to vector
Any vectors with an E.coRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
I2 cDNA libraries
30. Screening procedures
Screening
Colony and plaque hybridization
Expression screening
Hybrid arrest and release
Chromosome walking (repeat screening)
Gene libraries and screening
31. Screening
The process of identifying one particular
clone containing the gene of interest from
among the very large number of others in the
gene library .
1. Using nucleic acid probe to screen the library
based on hybridization with nucleic acids.
2. Analyze the protein product.
I3 Screening procedures
32. Screening libraries
Hybridization to identify the interested DNA or
its RNA product
1. Radiolabeled probes which is complementary to a
region of the interested gene
Probes:
• An oligonucleotide derived from the sequence
of a protein product of the gene
• A DNA fragment/oligo from a related gene of
another species
2. Blotting the DNA or RNA on a membrane
3. Hybridize the labeled probe with DNA membrane
(Southern) or RNA (Northern) membrane
Searching the genes of interest in a DNA library
I3 Screening procedures
33. Colony and plaque hybridization
Transfer the DNA in the plaque or colony to a
Nylon or nitrocellulose membrane
Phage DNA bind to
the membrane directly
Bacterial colonies must be lysed to
release DNA on the membrane
surface.
Hybridization (in a solution
Containing Nucleic acid probe)
Wash to remove unhybri-
dization probe and visualize
X-ray
film(radio-
actively
labeled )
antibody or
enzyme
(modified
nucleotide
labeled
Line up the hybridizated region or
repeated hybridization
(Alkali treatment)
I3 Screening procedures
34. Identify the protein product of an
interested gene
1. Protein activity
2. Western blotting using a specific
antibody
I3 Screening procedures
Expression screening
35. Expression screening
If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins. However,
this screening may miss the right clone
I3 Screening procedures
36. Expression screening
The procedure
‘Plaque lift’ ( taken by placing a
membrane on the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening
to isolate pure plaques
Antibodies can be used to screen the
expression library.
I3 Screening procedures
37. Hybrid arrest and screen
Individual cDNA clones or pools of clones can
be used to hybridize to mRNA preparation
Hybrid arrest :translate the mRNA population
directly, and the inhibition of translation of
some products detected.
Hybrid release translation : purify the
hybrids and the hybridized mRNAs released
from them and translated, it identifies the
protein encoded by the cDNA clone
I3 Screening procedures
38. I3-5 Chromosome walking
Definition: To clone the desired gene by
repeated isolating adjacent
genomic clones from the library.
to obtain overlapping genomic clones
that represent progressively longer
parts of a particular chromosome .
I3 Screening procedures
39. Process:
1. Prepare a probe from the end insert .
2.The probe are used to re-screen the library
by colony or plaque hybridization
3.Analyzed the new isolate clones and posited
them relative to the starting clone.
some will be overlapping.
4. Repeated the whole process using a probe
from the distal end of the second clone.
I3 Screening procedures
40. }
}
}
}
Vector arm Genomic clone insert Vector arm
Prepare probe from
ends of insert
Re-screen genomic
library
Restriction
Restriction map new
genomic clones
Prepare new probes from distal ends of least overlapping insert.
Re-screen genomic library . Restriction map new genomic clones
Chromosome walking