DNA manipulative enzymes
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This document summarizes different types of DNA manipulative enzymes including nucleases, ligases, polymerases, and modifying enzymes. It describes the functions of various nucleases like exonucleases and endonucleases that degrade DNA. Ligases repair breaks in double-stranded DNA. Polymerases make copies of DNA or RNA templates. Restriction endonucleases recognize and cut palindromic DNA sequences and are classified into types I, II, and III. The document also discusses how to convert blunt ends produced by restriction enzymes into sticky ends using linkers, adaptors, or homopolymer tailing.
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Restriction enzymes are enzymes that cut DNA at specific recognition sequences. There are three main types of restriction enzymes - Type I, II, and III. Type II restriction enzymes are the most commonly used in molecular cloning and DNA manipulation. They recognize short palindromic DNA sequences of 4-8 base pairs and cleave DNA within or nearby these recognition sites. Restriction enzymes have many applications including DNA sequencing, DNA libraries, and recombinant DNA techniques which enable genetic engineering.
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A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries will be discussed: genomic libraries and complementary DNA (cDNA) libraries.
Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome.
In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another.
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Chromosome walking is a method used to isolate and clone a particular gene or allele by using overlapping DNA fragments as probes to successively isolate flanking genomic clones. It involves constructing a genomic library, identifying a starting sequence fragment, using that as a probe to isolate an adjacent fragment, then using that new fragment as a probe to isolate the next overlapping fragment in a walking process. It has applications in analyzing genetically transmitted diseases and discovering single nucleotide polymorphisms, but can be slowed by repeated sequences and unclonable DNA regions.
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This document provides an overview of genome sequencing. It discusses that genome sequencing involves revealing the order of bases in an entire genome rather than sequencing genes one by one. Several methods of genome sequencing are described, including Sanger sequencing, automated sequencing, and ABE. Sanger sequencing was an early method that involved chain termination with dye-labeled dideoxynucleotides. Automated sequencing improved on this by running multiple reactions simultaneously in a single tube. Genome sequencing provides a wealth of genetic information and helps understand gene functions and interactions on a full genomic scale.
restriction enzymes
Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. They break the covalent bonds within a DNA strand and the hydrogen bonds between the strands. Bacteria use these enzymes to protect themselves from foreign DNA. Restriction enzymes are now used widely in laboratories to cut DNA in recombinant DNA techniques and other applications like DNA fingerprinting. They produce sticky or blunt ends that allow targeted joining of DNA fragments.
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Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
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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.
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This document discusses gene organization in microbes. It begins by defining the term "genome" and explaining that a genome contains an organism's complete set of DNA. Prokaryotes like bacteria have simpler genomes than eukaryotes, with a single circular chromosome and sometimes additional plasmids. Plasmids are small circular DNA structures that can be transferred between bacteria. The document then compares prokaryotic and eukaryotic chromosomes, noting that eukaryotes have membrane-bound nuclei while prokaryotes do not. It also discusses viral genomes, karyotypes, and different hypotheses about the relationship between genes and proteins.
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DNA manipulative enzymes can be grouped into four classes: nucleases, ligases, polymerases, and modifying enzymes. Nucleases break DNA phosphodiester bonds, including exonucleases which remove nucleotides from DNA ends and endonucleases which cleave internal bonds. Ligases seal DNA nicks using phosphodiester bonds. Polymerases synthesize new DNA strands using existing DNA or RNA as templates. Modifying enzymes alter DNA through addition or removal of chemical groups from nucleotides. These enzymes are essential tools for recombinant DNA technology.
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This presentation deals with DNA replication in mamalian mitochondria. Mammalian mtDNA is replicated by proteins distinct from those used for nuclear DNA replication. According to the strand displacement model, replication is initiated from two distinct origins, OH and OL.
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Polymerases,
M.Prasad Naidu discusses several types of polymerases including DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, and RNA-dependent RNA polymerases. Reich et al. and Baltimore and Franklin demonstrated RNA-primed RNA synthesis using the enzyme RNA-dependent RNA polymerase, also called RNA replicase. Spiegelman et al. isolated RNA replicase from bacteriophage Qβ which requires an RNA template, magnesium ions, and ribonucleoside triphosphates to function.
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DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro.
Genomic library construction
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.
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This document provides an overview of genome sequencing. It discusses that genome sequencing involves revealing the order of bases in an entire genome rather than sequencing genes one by one. Several methods of genome sequencing are described, including Sanger sequencing, automated sequencing, and ABE. Sanger sequencing was an early method that involved chain termination with dye-labeled dideoxynucleotides. Automated sequencing improved on this by running multiple reactions simultaneously in a single tube. Genome sequencing provides a wealth of genetic information and helps understand gene functions and interactions on a full genomic scale.
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Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. They break the covalent bonds within a DNA strand and the hydrogen bonds between the strands. Bacteria use these enzymes to protect themselves from foreign DNA. Restriction enzymes are now used widely in laboratories to cut DNA in recombinant DNA techniques and other applications like DNA fingerprinting. They produce sticky or blunt ends that allow targeted joining of DNA fragments.
Rolling Circle Replication ppt
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
transgene silencing
Transgene silencing refers to the downregulation or suppression of transgene expression in genetically modified organisms. There are several mechanisms by which this can occur, including RNA interference pathways, epigenetic effects, and position effects from chromosomal integration site. Post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) are two major types. PTGS affects mRNA levels after transcription, while TGS involves DNA/chromatin methylation and inhibits transcription. Virus-induced gene silencing (VIGS) uses recombinant viruses to trigger RNAi against plant genes, and is useful for functional analysis.
Recombinant DNA Technology
RDT,Gene cloning, Restriction Endonucleases, Blunt end,Sticky end, DNA ligase, Vestors, PCR, DNA Polymerase, Bloting techniques, Application of Recombinant technology, Genomic Library, cDNA Library
YEAST TWO HYBRID SYSTEM
Simply describe the yeast tow hybrid,where you can find definition ,principle,procedure,advantages and disadvantages .
Chromosome walking
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.
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The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
Transposons (2) (3)
Transposons are mobile genetic elements that can move within genomes. They were first discovered on plasmids carrying antibiotic resistance genes. It was found that resistance genes could move between plasmids via transposition. This explained how unrelated plasmids could acquire the same resistance genes. There are two classes of transposons - class I are "copy and paste" elements found in eukaryotes, while class II are "cut and paste" elements in prokaryotes. Many resistance plasmids have evolved rapidly by acquiring additional genes via transposon movement within and between plasmids and chromosomes. Composite transposons contain insertion sequence elements flanking resistance genes. Larger transposons are built up by inte
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This document discusses the mechanisms that ensure high fidelity in the transfer of genetic information from DNA to RNA to proteins. It describes how DNA replication, transcription, and translation rely on base complementarity and how the molecular machines that perform these processes, such as DNA polymerase and RNA polymerase, have evolved to select for correct Watson-Crick base pairs through monitoring their distinct geometry. It also discusses how aminoacyl-tRNA synthetases attach the correct amino acid to tRNAs with high selectivity using a "double-sieve" mechanism and how tRNA selection in the ribosome exploits kinetic proofreading to further increase fidelity.
Nucleases
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 replication in eukaryotes
DNA replication is the process by which a cell makes an identical copy of its DNA. It occurs in three main steps: initiation, elongation, and termination. Initiation begins at origins of replication and involves unwinding of the DNA double helix by helicases. During elongation, DNA polymerases add nucleotides to build new strands based on the existing DNA templates. Termination occurs when the replication forks meet at the end of the DNA molecule. DNA replication is semi-conservative, meaning each new DNA molecule contains one original and one new strand of DNA.
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DNA manipulative enzymes
- 1. DNA manipulative enzymes Dr. Mayank Chaudhary Assistant Professor Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala, Haryana
- 2. • Classification based on type of reaction catalyzed: 1) Nucleases: Cut/degrade nucleic acids 2) Ligases: Join nucleic acid molecules 3) Polymerases: Make copies of molecules 4) Modifying enzymes: Remove/add chemical groups
- 3. Nucleases • Degrade DNA molecules by breaking phosphodiester bonds that link one nucleotide to the next in a DNA strand. Types: 1) Exonuclease: Remove nucleotides from the end of DNA molecule. 2) Endonuclease: Breaks internal phosphodiester bonds within DNA molecule.
- 7. Ligases • Repair single-stranded breaks that arise in double stranded DNA molecules during replication. • Can also join two individual fragments of double stranded DNA. • Catalyzes covalent closure of nicks (missing phosphodiester linkages, no missing bases) by using energy from NAD or ATP
- 8. Polymerases • Enzymes that synthesize new strand of DNA complementary to an existing DNA or RNA template. Types: • DNA pol I: Isolated from E.coli and attaches to short, single stranded region mainly in double stranded DNA molecule. It then synthesizes complementary new strand degrading existing strand as it proceeds. • Klenow fragment: Modified DNA pol I that retains polymerase activity but no nuclease activity due to removal of segment. • DNA pol I from bacterium Thermus aquaticus is the Taq DNA polymerase used in PCR. • Reverse Transcriptase: Uses RNA as template for synthesis of cDNA.
- 10. DNA modifying Enzymes 1) Alkaline Phosphatase: Removes phosphate group present at 5’ end of DNA molecule. 2) Polynucleotide Kinase: Adds phosphate group onto 5’ end. 3) Terminal deoxynucleotidyl transferase: Adds one or more dNTPs onto 3’ end of DNA molecule.
- 12. Restriction Endonucleases (REs) • Discovered in strains of bacteria that were immune to bacteriophage infection due to phenomenon called as Host-controlled restriction. • First RE to be discovered: HindII by Nathans, Arber and Smith.
- 13. • Nomenclature: Classification of REs: Type I: Possess both restriction and modification activity. Cleavage occurs 1000bp away from recognition site. Type II: Restriction and modification activities are mediated by separate enzymes. Recognizes palindromic sequence and cleavage occurs at restriction site. Type III: Recognition and methylation of DNA sequence and cleavage at 24-26bp away.
- 14. Number of restriction sites/recognition sequences for particular RE in a DNA molecule: 4n (n= length of recognition sequence) Eg. For GATC recognition sequence: 44=256 nucleotides For GGATTC recognition sequence: 46=4096 nucleotides
- 15. • Blunt ends/flush ends: Double stranded cut in the middle of recognition sequence. Eg. PvuII and AluI are blunt end cutters. • Sticky/cohesive ends: Two DNA strands are not cut at exactly same position and cleavage is staggered/ Eg. NotI and EcoRI. It is easier for DNA ligase to work on sticky ends due to hydrogen bonding between complementary bases.
- 16. How Blunt ends can be converted to Sticky ends?? 1) Linkers 2) Adaptors 3) Homopolymer tailing Linkers: Blunt-ended double stranded DNA molecules containing restriction site.
- 17. Adaptors: Short synthetic oligonucleotide similar to linkers but is pre-treated to have one sticky end.
- 18. Homopolymer tailing: Series of nucleotides is attached to 3’-OH termini of double stranded DNA molecule with help of enzyme terminal deoxynucleotidyl transferase.
- 19. References • T. A. Brown. Gene cloning & DNA analysis-An Introduction. Seventh edition.