Cosmids.pptx
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A cosmid is a type of plasmid vector that can accommodate large DNA fragments of up to 45 kb. It contains cos sites from bacteriophage lambda that allow for in vitro packaging into phage particles. Cosmids are used to create gene libraries and clone large genes due to their ability to carry larger DNA fragments compared to plasmids. They are constructed by joining linearized plasmid DNA with DNA fragments containing cos sites from bacteriophage like P1.
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Library screening
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.
Chromosome walking
Chromosome walking is a technique used to analyze large regions of DNA by identifying overlapping clones. It involves selecting a starting clone, subcloning fragments from the ends, and using those fragments to screen a genomic library and identify neighboring clones with overlapping sequences. This process is repeated to "walk" along the chromosome. Chromosome walking was developed in the 1980s and is useful for finding mutations associated with genetic diseases and detecting single nucleotide polymorphisms, though it has limitations with repetitive sequences.
lambda cloning vector
- Lambda bacteriophage cloning vectors were developed to overcome limitations in the size of DNA that could be inserted into unmodified lambda vectors.
- Segments of the non-essential lambda genome could be deleted to allow insertion of up to 18kb of new DNA while still allowing packaging.
- Natural selection was used to generate lambda strains lacking restriction sites, allowing restriction-based cloning.
- The first lambda vectors were insertion and replacement vectors, while later cosmids allowed cloning of fragments up to 52kb.
Finding ORF
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
Cosmid
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Artificial Vectors
Artificial vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), P1-derived artificial chromosomes (PACs), and human artificial chromosomes (HACs) can carry large DNA fragments for research purposes. BACs can hold up to 300kb of DNA and are used to sequence genomes. YACs can carry 500kb of DNA but are prone to rearrangement. HACs are separate microchromosomes that act as new chromosomes and can carry therapeutic genes for research including disease models.
Microinjection
a proper description about the process microinjection and also about gene transfer. and different types of DNA delivery methods.
with advantages, disadvantages, limitations and applications.
Cell disruption
Cell disruption is the process of breaking open cell walls to extract intracellular fluid and components without damaging them. The goal is an effective disruption while keeping products active. Methods include mechanical techniques like bead beating, blending, and homogenization which use physical force. Non-mechanical techniques involve freeze-thawing, osmotic shock, chemicals, enzymes, or electricity to disrupt cell walls and membranes in different ways. The optimal method depends on cell type and desired outcome.
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Library screening
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.
Chromosome walking
Chromosome walking is a technique used to analyze large regions of DNA by identifying overlapping clones. It involves selecting a starting clone, subcloning fragments from the ends, and using those fragments to screen a genomic library and identify neighboring clones with overlapping sequences. This process is repeated to "walk" along the chromosome. Chromosome walking was developed in the 1980s and is useful for finding mutations associated with genetic diseases and detecting single nucleotide polymorphisms, though it has limitations with repetitive sequences.
lambda cloning vector
- Lambda bacteriophage cloning vectors were developed to overcome limitations in the size of DNA that could be inserted into unmodified lambda vectors.
- Segments of the non-essential lambda genome could be deleted to allow insertion of up to 18kb of new DNA while still allowing packaging.
- Natural selection was used to generate lambda strains lacking restriction sites, allowing restriction-based cloning.
- The first lambda vectors were insertion and replacement vectors, while later cosmids allowed cloning of fragments up to 52kb.
Finding ORF
Open reading frame is part of reading frame that contains no stop codons or region of amino acids coding triple codons.
ORF starts with start codon and ends at stop codon.
Cosmid
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Artificial Vectors
Artificial vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), P1-derived artificial chromosomes (PACs), and human artificial chromosomes (HACs) can carry large DNA fragments for research purposes. BACs can hold up to 300kb of DNA and are used to sequence genomes. YACs can carry 500kb of DNA but are prone to rearrangement. HACs are separate microchromosomes that act as new chromosomes and can carry therapeutic genes for research including disease models.
Microinjection
a proper description about the process microinjection and also about gene transfer. and different types of DNA delivery methods.
with advantages, disadvantages, limitations and applications.
Cell disruption
Cell disruption is the process of breaking open cell walls to extract intracellular fluid and components without damaging them. The goal is an effective disruption while keeping products active. Methods include mechanical techniques like bead beating, blending, and homogenization which use physical force. Non-mechanical techniques involve freeze-thawing, osmotic shock, chemicals, enzymes, or electricity to disrupt cell walls and membranes in different ways. The optimal method depends on cell type and desired outcome.
P br322
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
Gen bank databases
GenBank is a database that contains annotated nucleotide and protein sequences. It includes genomic DNA, mRNA, and EST sequences. There are three main sections in a GenBank file - the header, features, and sequence. The header provides definition, accession number, organism, and reference information. The features section contains gene and protein annotation. The sequence section displays the actual nucleotide or amino acid sequence. Understanding the GenBank file format helps effectively search and retrieve sequences from this important biological database.
Ligase enzyme
DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between DNA fragments, joining two DNA strands together. It plays an important role in DNA replication by joining Okazaki fragments and filling in gaps, as well as in DNA repair and genetic engineering techniques like cloning. The most commonly used DNA ligase is from bacteriophage T4, which utilizes ATP as a cofactor and works efficiently at lower temperatures to ligate DNA strands with either sticky or blunt ends.
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Physiochemical properties of dna
This document discusses several physiochemical properties of DNA including:
- Absorption of UV light at 260nm, which can be used to determine DNA concentration. Double stranded DNA absorbs less light than single stranded DNA.
- Buoyant density of 1.73 g/cm3 in cesium chloride, which can estimate GC content through centrifugation separation. Higher GC DNA migrates lower.
- Denaturation of the double helix into single strands above 90°C or through chemical means like low pH or urea, monitored by increased UV absorption.
- Renaturation of single strands back into the double helix upon cooling through hydrogen bonding of complementary bases.
Transformation and transfection
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
Genomic and c dna library
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.
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Cosmids vector
1. A cosmid is a type of plasmid vector that contains sequences from bacteriophage lambda, specifically the cos sites. This allows DNA fragments up to 45kb to be packaged into phage particles and transduced into bacteria.
2. Cosmids are developed by combining features of plasmid and phage vectors. They can accept large DNA inserts through packaging and transduction while also replicating stably inside bacteria like plasmids.
3. Cloning with cosmids involves inserting DNA fragments into the cosmid, ligating to form concatemers, in vitro packaging into phage particles, and transducing the particles into bacteria where the cosmids replicate as plasmids.
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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.
Complementary DNA (cDNA) Libraries
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.
PLANT TRANSFORMATIVE VECTORS.pptx
Plant transformation vectors can be classified into cloning vectors, expression vectors, and integration vectors. Cloning vectors are small DNA molecules used to insert, store, and manipulate foreign DNA. Common cloning vectors include plasmids, bacteriophages, cosmids, and BACs/YACs. Expression vectors allow foreign DNA to be inserted and expressed in host cells. The Ti plasmid is often used as a plant expression vector due to genes that mediate DNA transfer to plant cells. Vector choice depends on desired DNA insert size, host system, and purpose of cloning/expression.
PLANT TRANSFORMATIVE VECTORS.pptx
Plant transformation, vectors, vectors classification, cloning vectors, types of cloning vectors, plasmid vectors, classification of plasmid vectors, pBR322, advantages and disadvantages of vectors, Bacteriophage vectors, phage Lambda, M13 vectors, cosmids, bacterial artificial chromosome, yeast artificial chromosome, shuttle vectors, expression vectors, Ti plasmid, its advantages and disadvantage, conclusion
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Gen bank databases
GenBank is a database that contains annotated nucleotide and protein sequences. It includes genomic DNA, mRNA, and EST sequences. There are three main sections in a GenBank file - the header, features, and sequence. The header provides definition, accession number, organism, and reference information. The features section contains gene and protein annotation. The sequence section displays the actual nucleotide or amino acid sequence. Understanding the GenBank file format helps effectively search and retrieve sequences from this important biological database.
Ligase enzyme
DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between DNA fragments, joining two DNA strands together. It plays an important role in DNA replication by joining Okazaki fragments and filling in gaps, as well as in DNA repair and genetic engineering techniques like cloning. The most commonly used DNA ligase is from bacteriophage T4, which utilizes ATP as a cofactor and works efficiently at lower temperatures to ligate DNA strands with either sticky or blunt ends.
Expression vectors
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Physiochemical properties of dna
This document discusses several physiochemical properties of DNA including:
- Absorption of UV light at 260nm, which can be used to determine DNA concentration. Double stranded DNA absorbs less light than single stranded DNA.
- Buoyant density of 1.73 g/cm3 in cesium chloride, which can estimate GC content through centrifugation separation. Higher GC DNA migrates lower.
- Denaturation of the double helix into single strands above 90°C or through chemical means like low pH or urea, monitored by increased UV absorption.
- Renaturation of single strands back into the double helix upon cooling through hydrogen bonding of complementary bases.
Transformation and transfection
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
Genomic and c dna library
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.
Genome sequencing
The document discusses genome sequencing and related topics. It begins by defining what a genome is - the complete set of DNA in an organism. It then discusses the different types of genomes, such as prokaryotic and eukaryotic, including nuclear, mitochondrial, and chloroplast genomes. The document also defines genomics as the comprehensive study of whole genomes and all gene interactions, distinguishing it from traditional genetics which focuses on single genes. It outlines some key milestones in genomic sequencing and the technical foundations that enabled sequencing whole genomes. Finally, it describes the main approaches used for genome sequencing projects, including hierarchical shotgun sequencing and whole genome shotgun sequencing.
C dna
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Yeast cloning vectors allow DNA fragments to be replicated and expressed in yeast cells. There are several types of yeast vectors including integrating plasmids (YIps) that replicate by integrating into yeast chromosomes, episomal plasmids (YEps) that replicate independently but can also integrate, and replicating plasmids (YRps) that contain an autonomously replicating sequence (ARS) and replicate at low copy numbers. Yeast artificial chromosomes (YACs) are engineered chromosomes containing telomeric, centromeric, and ARS sequences that can clone very large DNA fragments of up to 3000 kb.
Histotypic culture
Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.
Isolation, purification and characterisation of protein
This document discusses strategies for isolating, purifying, and characterizing proteins. It describes various methods for extracting proteins from tissues or cells, such as cryogenic grinding, ultrasound homogenization, and lysis buffers. Purification techniques are then outlined, including precipitation, size exclusion chromatography, and isoelectric focusing. Finally, methods for identifying purified proteins are summarized, like mass spectrometry, N-terminal sequencing, and analyzing protein structure using techniques like circular dichroism spectroscopy and X-ray crystallography.
Restriction Mapping
Restriction enzymes cut DNA molecules at specific recognition sites. Restriction mapping involves digesting an unknown DNA segment with restriction enzymes and analyzing the fragment sizes to determine the locations of restriction sites. One method involves single and double digestions with two enzymes followed by gel electrophoresis to separate the fragments by size. By comparing the fragment patterns between single and double digestions, the positions of each restriction site can be mapped, generating a restriction map of the DNA segment. Restriction mapping was previously important for characterizing cloned DNA but is now easier using DNA sequencing, though analysis of restriction sites remains useful for comparing chromosomal organization between strains.
Chromosome walking jumping transposon tagging map based cloning
Chromosome walking, jumping, and transposon tagging are techniques used for gene mapping and cloning. Chromosome walking involves isolating overlapping DNA fragments in steps to characterize large chromosome regions. Chromosome jumping uses rare cutting enzymes to isolate larger DNA fragments spanning hundreds of kb. Transposon tagging involves inducing transposon insertion mutations, identifying the disrupted gene, and using the transposon as a tag to clone the gene. Map-based cloning localizes a gene of interest by identifying closely linked markers, screening libraries to find flanking markers, and identifying the gene between markers through complementation tests.
Lectut btn-202-ppt-l25. introduction of dna into host cells
This document discusses various methods for introducing DNA into host cells, which is an important step in genetic engineering. It describes transformation, which is introducing DNA into living cells, and transfection, which is introducing viral DNA into living cells. The document then outlines several biological, chemical, and physical methods for transformation and transfection in bacteria, plants, insects, and animal cells. These include techniques like bacterial infection with bacteriophage, Agrobacterium-mediated plant transformation, microprojectile bombardment, electroporation, microinjection, and calcium phosphate transfection. It also discusses factors that affect transformation efficiency.
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Abzymes, also known as catalytic antibodies, are monoclonal antibodies that exhibit enzymatic activity. They are able to bind to transition states of enzyme-catalyzed reactions with high specificity and affinity, stabilizing the transition state and increasing reaction rates. Abzymes can be artificially produced by immunizing animals with transition state analogs of reactions. They have potential applications in drug development, cancer treatment, and developing therapies for viral infections like HIV. Researchers have engineered an abzyme that can degrade an essential region of the HIV envelope protein, rendering the virus unable to infect cells.
Cosmids vector
1. A cosmid is a type of plasmid vector that contains sequences from bacteriophage lambda, specifically the cos sites. This allows DNA fragments up to 45kb to be packaged into phage particles and transduced into bacteria.
2. Cosmids are developed by combining features of plasmid and phage vectors. They can accept large DNA inserts through packaging and transduction while also replicating stably inside bacteria like plasmids.
3. Cloning with cosmids involves inserting DNA fragments into the cosmid, ligating to form concatemers, in vitro packaging into phage particles, and transducing the particles into bacteria where the cosmids replicate as plasmids.
Library screening
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.
Complementary DNA (cDNA) Libraries
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.
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PLANT TRANSFORMATIVE VECTORS.pptx
Plant transformation, vectors, vectors classification, cloning vectors, types of cloning vectors, plasmid vectors, classification of plasmid vectors, pBR322, advantages and disadvantages of vectors, Bacteriophage vectors, phage Lambda, M13 vectors, cosmids, bacterial artificial chromosome, yeast artificial chromosome, shuttle vectors, expression vectors, Ti plasmid, its advantages and disadvantage, conclusion
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cloning vectors.pptx Biotechnology class
A cloning vector is a small piece of DNA, such as a plasmid, virus, or artificial chromosome, that can accept foreign DNA and be replicated within a host cell. The summary describes the main types of cloning vectors, including plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. It also outlines some key features of cloning vectors like origins of replication, cloning sites, and selectable markers. The choice of vector depends on factors like the size of the DNA insert and cloning efficiency.
Cloning vectors
- Lambda bacteriophage cloning vectors were developed to overcome limitations in the size of DNA that could be inserted into unmodified lambda vectors.
- Segments of the non-essential lambda genome could be deleted to allow insertion of up to 18kb of new DNA while still allowing packaging.
- Natural selection was used to generate lambda strains lacking restriction sites, allowing restriction-based cloning.
- The first lambda vectors were insertion and replacement vectors, while later cosmids allowed cloning of fragments up to 52kb.
Cloning vector
This document discusses cloning vectors, which are DNA molecules used to transport cloned DNA sequences between biological hosts. It defines a cloning vector as a small piece of DNA from a virus, plasmid, or cell that can maintain foreign DNA for cloning. The summary describes the key features of cloning vectors, including an origin of replication, cloning site, selectable marker, and optional reporter gene. It also lists common vector types like plasmids, bacteriophages, cosmids, and artificial chromosomes, and factors that determine the choice of vector, such as insert size.
Cloning vectors
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Cosmid Vectors, YAC and BAC Expression Vectors
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
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Recombinant DNA technology involves combining DNA from two different organisms and inserting it into a host. This is done by using restriction enzymes to cut the DNA into fragments, which are then inserted into cloning vectors like plasmids, bacteriophages, or artificial chromosomes. The recombinant DNA is then inserted into a host organism using techniques like transformation or transfection. Gel electrophoresis can be used to analyze the results and identify successful recombinant clones. While cloning has potential medical applications, reproductive cloning of humans remains unsafe and controversial.
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Vectors are DNA molecules that transport foreign DNA into host cells. Common vectors include plasmids, bacteriophages, cosmids, and artificial chromosomes. Cloning vectors are used to replicate and express foreign DNA in host organisms. Restriction enzymes cut DNA at specific recognition sites, while DNA ligase joins cut DNA fragments together. These tools enable cloning of DNA fragments into vectors for amplification and study.
cloning vectors.pptx Biotechnology Notes
A cloning vector is a small piece of DNA, such as a plasmid, virus, or artificial chromosome, that can accept foreign DNA and be used to clone that DNA and replicate it in a host cell. This document discusses the history and features of common cloning vectors like plasmids, bacteriophages, cosmids, and artificial chromosomes. It explains how vectors are chosen based on factors like insert size and used in molecular cloning by digesting DNA with restriction enzymes, ligating into the vector, transforming into host cells, and selecting for recombinant clones.
Cloning vectors.pptx
Cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism. The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs).
unit-2 cloning vector, r-DNA Technology, PCR.pptx
Cloning vectors are small pieces of DNA that can be stably maintained in an organism and have foreign DNA inserted into them for cloning purposes. The most commonly used cloning vectors are genetically engineered plasmids. Plasmids are taken from bacteria and can replicate within bacterial cells. Other types of cloning vectors include bacteriophages, cosmids, yeast artificial chromosomes, and bacterial artificial chromosomes, which can accommodate larger DNA fragments. Restriction enzymes and DNA ligase are used to cut and join DNA fragments for cloning into vectors.
Lecture_3-Vectors.pdf
The document discusses cloning vectors. It describes what a cloning vector is, including that it is a small piece of DNA that can stably maintain foreign DNA for cloning purposes. Common types of cloning vectors are described in detail, including plasmids, bacteriophages, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes, and plant virus vectors. Key features of cloning vectors like origins of replication, antibiotic resistance genes, and cloning sites are also summarized.
cloning vectors.ppt
Cloning vectors are small DNA molecules that can accept foreign DNA inserts and replicate within a host cell. They contain features like an origin of replication, antibiotic resistance genes, and restriction enzyme cleavage sites. Common vector types include plasmids, bacteriophages, cosmids, and artificial chromosomes. The choice of vector depends on factors like the size of the DNA insert and the intended use. Vectors allow amplification and manipulation of cloned DNA fragments.
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- 2. Cos sites + Plasmid = Cosmids • A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid with a bacterial “ori” sequence and a “cos” sequences derived from the lambda phage. • It can be used as gene-cloning vectors in conjunction with the in vitro packaging system. • For example, the commonly used cosmid pLFR-5 has two cos sites (cos ends) from bacteriophage λ.
- 5. • Antibiotic-resistant genes present • Cohesive end sites present • Size of DNA fragments that can be accommodated is up to 45 kb • Cos sites present • It contains 37-52 kb of DNA, based on the size of bacteriophage packaging • It also comprises a gene for selection frequently for transformed cells to be identified on a medium with the antibiotic
- 6. • Cosmid pick up relatively larger DNA fragments than the plasmid • As cosmids pick up large DNA fragments, they are used to establish gene libraries • Gene cloning through cosmids helps in the study of non-sence sequences in the genome of organisms. • Some cosmids are constructed by joining a linearized plasmid DNA with DNA fragments of p1 bacteriophage that have cos-ends. The P1 bacteriophage has the genome of 115 kbp. So, a DNA of 85 kbp can be packaged into the head of P1 phage. These cosmids help to clone large genes and gene clusters in bacteria.
- 7. • The packaging enzyme fails to pack recombinant cosmids into the phage head, if any one of the two cos-ends is missing. • Sometimes more than one recombinant cosmid join together to form a large DNA. If so, the packaging enzyme fails to pack the DNA into the phage head. • Slower replication • Higher frequency of recombination inside bacterial host. • Unstable inside E.coli host and thus easy to lose vector.
- 9. The following techniques and materials have been used for the project.
- 11. • http://vidyamandira.ac.in/pdfs/e_learning/ds_mi crobio/COSMID%20VECTORS%20MCBA%20P7%2 0T.pdf • Molecular Biotechnology - Principles and Applications of Recombinant DNA,Glick, B.R. and Pasternak • Principles of Gene Manipulation and Genomics- Primrose S. B. and Twyman R.M. • https://byjus.com/neet/difference-between- cosmid-and-phagemid/