Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Molecular cloning vectors

12,685 views

Published on

The molecular cloning vectors used in genetic engineering-plasmids,phage vectors,Hbrid vectors and artificial chromosomes

Published in: Education
  • DOWNLOAD FULL BOOKS, INTO AVAILABLE FORMAT ......................................................................................................................... ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y8nn3gmc } ......................................................................................................................... ......................................................................................................................... ......................................................................................................................... .............. Browse by Genre Available eBooks ......................................................................................................................... Art, Biography, Business, Chick Lit, Children's, Christian, Classics, Comics, Contemporary, Cookbooks, Crime, Ebooks, Fantasy, Fiction, Graphic Novels, Historical Fiction, History, Horror, Humor And Comedy, Manga, Memoir, Music, Mystery, Non Fiction, Paranormal, Philosophy, Poetry, Psychology, Religion, Romance, Science, Science Fiction, Self Help, Suspense, Spirituality, Sports, Thriller, Travel, Young Adult,
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here

Molecular cloning vectors

  1. 1. MOLECULAR CLONING VECTORS Presented By, Sonia John II M.Sc Zoology
  2. 2. 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.
  3. 3. DIFFERENT TYPES • PLASMIDS • PHAGES • HYBRID VECTORS • ARTIFICIAL CHROMOSOMES
  4. 4. 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
  5. 5. • Chang and Cohen first proved the use of plasmid as gene cloning vectors. • CHARACTERISTCS OF IDEAL PLASMID VECTORS 1. Small size: 1.2-3kb 2. Copy number 3. Genetic marker 4. Origin of replication 5. Unique Restriction Site 6. No pathogenicity 7. Multiple Cloning Site 8. Promoter Sequence
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. • 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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;
  15. 15. 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
  16. 16. 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
  17. 17. 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 plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome
  18. 18. • 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
  19. 19. 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
  20. 20. 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
  21. 21. Other types • Expression vectors • Shuttle vectors • Integration Vectors • Transposons,Retroviruses,Adenoviruses,Baculo viruses
  22. 22. 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
  23. 23. REFERENCES 1. 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

×