Molecular cloning vectors
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Transcript of Molecular cloning vectors
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MOLECULAR CLONING VECTORSPresented By,Sonia JohnII M.Sc Zoology
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CLONING VECTORS• DNA molecules that can carry a foreign DNA
fragment when inserted into it
• Share four common properties:
1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for selection.
3. Unique restriction sites to facilitate cloning of insert DNA.
4. Minimum amount of nonessential DNA to optimize cloning.
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DIFFERENT TYPES•PLASMIDS
•PHAGES
•HYBRID VECTORS
•ARTIFICIAL CHROMOSOMES
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PLASMIDS• Extra-chromosomal,self-replicating ,double stranded,
closed circular DNA molecules present in the bacterial cell
• Copy Number: High copy number and low
copy number
• Small size
• No of genes is limited
• Episomes
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• Chang and Cohen first proved the use of plasmid as gene cloning vectors.
• CHARACTERISTCS OF IDEAL PLASMID VECTORS1. Small size: 1.2-3kb2. Copy number3. Genetic marker4. Origin of replication5. Unique Restriction Site6. No pathogenicity7. Multiple Cloning Site8. Promoter Sequence
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NATURAL AND ARTIFICIAL PLAMIDS• Plasmids isolated from bacteria&directly used for gene
cloning without any modification :-Natural Plasmid
• But not used in gene cloning due to large size,confer pathogenicity etc
• Artificial/derived plasmids constructed by cutting out unwanted portions from wild type plasmids.Eg:pBR322
• These are of much use in gene transfer
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ADVANTAGES AND DISADVANTAGES• ADVANTAGES:
1. Readily isolated from cells
2. Can be reintroduced into a bacterial cell
3. Possess a single restriction site for 1 or more restriction enzyme
4. MCS
5. Introduction of a linear molecule does not alter its replication
• DISADVANTAGES
1. Cannot accept large fragments
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LAMBDA PHAGE VECTORS• Lambda phage is a bacterial virus that infects E.coli.
• Consists of an icosahedral head and flexible tail.
• Phage DNA packed inside the head
• Lambda DNA is a linear DNA duplex with cohesive single strand extensions
• The single stranded extensions of the DNA are COMPLEMENTARY to each other and consists of 12 nucleotides:: Cos Sites
• Free end of the cos site has a 5’ phosphate group
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• Lambda Phage DNA can carry large DNA fragments and integrate into host chromosome
• Wild type Lambda DNA can carry only small fragments;The unwanted seq are cut out:- CONSTRUCTED LAMBDA DNA VECTOR
• It is of 2 types:
1. Insertion Vectors- 1 restriction site;fragments upto 18kb
2. Replacement Vectors-2 restriction sites;Removal of stuffer seq
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ADVANTAGES & DISADVANTAGES• ADVANTAGES
1. Large DNA fragments upto 23kb can be carried
2. Efficiency of gene transfer is high
• DISADVANTAGES
1. Rec.DNA may enter lytic cycle
2. Lambda phage has narrow host range
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M13 BACTERIOPHAGE VECTOR• A single stranded DNA contaning phage virus;Can
infect F+ cells
• Consists of about 6402 b.p
• The Intergenic Seq (IS) of M13 modified so as to introduce additional restriction site
• The gene LacZ is integrated and then introduced into the IS to get M13 Vector
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HYBRID VECTORS:Cosmids and Phagemids
COSMIDS• Artificial plasmid containing Cos-sites of lambda DNA
• Formed by joining ends of a linearised plasmid DNA with cos-sites
• Has an ori,selectable marker,cloning sites of plasmid DNA
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Features fo COSMID• Circular,ds DNA
• 2 complementary ss regions- cos sites
• Cosmid DNA does not code for phage protein and host cell lysis
• Does not involve in multiplication of phage particles
• Has an ori from plasmid- for independent repl.
• Has selectable marker gene & gene cloning site of plasmid DNA
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ADVANTAGES AND DISADVANTAGES• ADVANTAGES
1. Large DNA fragments
2. Used to establish gene libraries of lower &higher organisms
3. Gene cloning through cosmid helps in the study of non-sense seq in the genome of organism
• DISADVANTAGES
1. The packaging enzyme fails to pack rec cosmid into phage heads if 1 of cos-sites is missing;
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PHAGEMIDS• A.k.a Phasmids
• A hybrid vector that has origin of repl from a a plasmid and a lambda phage DNA
• It is constructed by inserting a linearised plasmid DNA into a cleaved lambda DNA
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ADVANTAGES• PHAGEMIDS can be maintained as plamids or phage
particles in E.coli
• The desired gene can be integrated into chromosomal DNA of E.coli using phagemids
• Plasmid portion can be released free from a phagemid after rec.phagemid is introduced into an E.coli Strain
• The phages having rec.phagemids can be stored easily for a long time
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ARTIFICIAL CHROMOSOMES• YEAST ARTIFICIAL CHROMOSOME(YAC)
• Purpose:• Cloning vehicles that propogate in eukaryotic cell hosts as
eukaryotic Chromosomes
• Clone very large inserts of DNA: 100 kb - 10 Mb
• Features:
YAC cloning vehicles are plasmidsFinal chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome
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• Often have a selection for an insert
• YAC cloning vehicles often have a bacterial origin of DNA replication (ori) and a selection marker for propogation of the YAC through bacteria.
• The YAC can use both yeast and bacteria as a host
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BAC AND PAC• BACs - Bacterial Artificial
Chromosomes• These chimeric DNA
molecules use a naturally-occurring low-copy number bacterial plasmid origin of replication, such as that of F-plasmid in E. coli.
• Can be cloned as a plasmid in a bacterial host, and its natural stability generally permits cloning of large pieces of insert DNA, i.e. up to a few hundred kb of DNA.
• PACs - P1-derived Artificial Chromosomes
• E. coli bacteriophage P1 is similar to phage lambda in that it can exist in E. coli in a prophage state.
• Exists in the E. coli cell as a plasmid, NOT integrated into the E. coli chromosome.
• P1 cloning vehicles have been constructed that permit cloning of large DNA fragments- few hundred kb of DNA
• Cloning and propogation of the chimeric DNA as a P1 plasmid inside E. coli cells
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Human Artificial Chromosome(HAC)• Synthetically produced vector DNA possessing the
characteristics of human chromosome.
• Discovered in 1997 by H.Williard
Advantages:
1. Can carry Human genes that are too long
2. Can carry genes to be introduced into the cells via gene therapy
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Other types•Expression vectors•Shuttle vectors•Integration Vectors•Transposons,Retroviruses,Adenoviruses,B
aculo viruses
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CONCLUSION• There are different types of cloning vectors used in
Genetic Engineering
• These include: Plasmids, Phages,Hybrid Vectors and Artificial Chromosome
• The best vector is chosen for use according to the purpose of use and according to how large/short the DNA fragment to be carried is
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REFERENCES1. Kumaresan.V. Bitotechnology.2001.Saras Publications
2. Rastogi.S.C. Biotechnology:Principles and Applications. Narosa Publishing House
3. Sasidhara.R. Animal Biotechnology.MJP Publications
4. Satyanarayana.U. Biotechnology . Books and Allied (P)Ltd.
5. http://en.wikipedia.org
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