Tools used in genetic engineering_ biotechnology

discuss about various tools used in genetic engineering or recombinant DNA technology.

1. Tools used in Genetic Engineering Prepared by: MS. Diptee gupta Assistant professor Krishna institute of pharmacy & Sciences, Kanpur 1

2. Cloning Vector Vectors are DNA molecules which can carry a foreign DNA fragment to be cloned. They are self replicating in an appropriate host cell. Any extra-chromosomal small genome is used as a vector. Eg : Plasmid, Bacteriophage, Cosmid, Yeast, Shuttle, Expression vector etc. Characteristics: Small in size Have a single restriction endonuclease site Have an origin of replication (Ori) Have 1-2 suitable marker genes , allow easy selection of transformed cells. Should easily isolated from cells. 2

3. 1. Plasmid vector These are extra- choromosomal , double stranded, circular, self replicating DNA molecules present in bacterial cell. They are widely used as as cloning vehicles. Almost all the bacteria have plasmids containing a low copy of number (1-4 per cells) or a high copy number (10 -100per cells). The size of plasmid varies from 1-500 kbp (kilo base pair). Eg : pBR322, pBR325, pUC19, pBR329, pMB9, pRK2501 3

4. pBR322 Vector: It is one of the artificial vector & most widely used plasmid from Escherichia coli plasmid. It have 4362 basepair (bp) in length. In the name of pBR322 P stands for Plasmid, BR stands for scientist name Bolivar & Rodriguez. The structure of pBR322 have: Origin of replication (Ori) Restriction enzyme site : almost 20 different restriction sites present in which 11 sites present in Tetracycline resistant region while 9 present in Ampicillin resistant region. Eg : Bam HI, HIND III, EcoR IV 4

5. Genetic markers: 2 selectable markers present – Ampicillin resistant site i .e the ampicillin gene codes for β-lactamase, which can be used for screening microorganisms when a foreign DNA is being inserted in the plasmid. Tetracycline resistance site – this gene degrades the antibiotic tetracycline and can be used for screening microorganisms. 5

6. 2. Bacteriophage vector: These are those virus which can replicate within bacteria . These vectors can accept short fragments of foreign DNA but they can carry larger segment than plasmid. Bacteriophage of E.coli: λ , Bacteriophage M13 & Fd . These vectors can carry larger segment over 25kbp as compare to plasmid vector. 6

7. 3. Cosmid vectors: (cos site + Plasmid) These are special type hybrid vectors that carry characteristics of both plasmid and bacteriophage vectors ( λ ) . They can carry upto 45 kb long DNA segment and can replicate as plasmid if they have suitable Ori. These can also packed in Phage capsid that allow the foreign gene to be transferred into cell by transduction. Some artificial chromosome vectors: Human artificial chromosome (HAC) Yeast artificial chromosome (YAC) Bacterial artificial chromosome (BAC) Shuttle vector 7

8. Yeast artificial chromosome (YAC): These are expression vectors that allow transcription and translation of inserted section of DNA. But these vectors usually produce chimeric effect, that make them less stable as compare to BAC. Shuttle vector: It is a plasmid vector but designed to replicate in 2 host cells such as E.coli & Streptomyces species. Generally the Ori of 2 vectors, combine in one vector to form a shuttle vector. 8

9. Restriction Endonuclease These are one of the most important group of enzymes used for manipulation of DNA. These are bacterial enzyme that can cut/split at specific nucleotide site ( Palindromic sequence ) and also make cut on plasmid and desired DNA. The target site for cutting may vary from one enzyme to another enzyme. These are also known as biological knives or biological scissors and belong to class of enzymes called Nucleases. HIND II first restriction enzyme to be isolated. 9

10. Nomenclature: named by a standard procedure— First letter- indicate genus Next 2 nd and 3 rd letter- indicate species 4 th letter – strain of organisms Roman number- indicate the order isolated from strain Eg ; Eco RI Escherichia (E), Coli (C), Strain – RY 13 (R), first endonuclease (I) to be isolated from strain Hind III Haemophilus (H), influenzae (in), Strain – Rd (d) , third endonuclease enzyme 10

11. Types : There are 3 main types of Restriction Endonuclease- Type I , Type II, Typer III. These enzymes differ from each other in their mode of action . Type I Type II Type III Consist of 3 different subunits 2 Identical subunit 2 different subunit Require ATP, Mg++, S- adenosyl methionine for restriction Require Mg++ for restriction Require ATP, Mg++, S- adenosyl Cleaves DNA at random site upto 1000bp away from recognition sequence Cleaves within recognition sequence Cleaves DNA about 25bp from recognition sequence Not used in rDNA Used in rDNA technology Not used Eg : Eco K I, Eco BI Eg ; Eco RI, Alu I Eg : Pst I, Hinf III 11

12. Mechanism of Restriction Endonuclease These enzymes cut / cleave the DNA molecule in 2 different ways- 1 .Blunt end / Flush end- when enzyme cleave both strand of DNA at the same point within recognition sequence, blunt ends are generated. Eg : Puv II, Hae III 12

13. 2 . Sticky end: In this , 2 strands of DNA are cut at different points, it generate protruding ends i.e. one strands of double helix extends a few base beyond the other strand. Eg ; Eco RI 13

14. DNA Ligase: It is a type of enzyme that facilitate the joining of DNA strands together by catalyzing the formation of phosphodiester bond . This phosphodiester bond is formed between phosphate group of 5` carbon of one deoxyribose with the –OH group at 3` carbon of another deoxyribose. Originally isolated from viruses but occur in E.coli & eukaryotic cells. Types: T4 DNA ligase : require ATP as cofactor & have the ability to join the blunt end of DNA fragments. E.Coli DNA ligase: require NAD+ as a cofactor & have the ability to join the sticky ends produced by restriction enzymes 14

15. DNA ligase enzymes participated in cellular DNA repair process and also in DNA replication process. It have extensive use in molecular biology laboratories for recombinant DNA experiments. 15