1. Recombinant DNA Technology Restriction Endonucleases; cloning and Transformation

2. Restriction Endonucleases Restriction endonucleases RESTRICT viruses Viral genome is destroyed upon entry Restriction endonuclease = Restriction enzymes Endo (inside), nuclease (cuts nucleic acid) Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside. The specific DNA sequence is called recognition sequence 1952-53: Luria and Human discovered the phenomenon of restriction and modification Named as host-induced, or host-controlled, variation

3. Few Restriction Enzymes EnzymeOrganism from which derived Target sequence (cut at *) 5' -->3' Bam HIBacillus amyloliquefaciensG* G A T C C Eco RIEscherichia coli RY 13G* A A T T C Hind IIIHaemophilus inflenzae RdA* A G C T T Mbo IMoraxella bovis*G A T C Pst IProvidencia stuartiiC T G C A * G Sma ISerratia marcescensC C C * G G G Taq IThermophilus aquaticusT * C G A Xma IXanthamonas malvacearumC * C C G G G

4. Protection of Self-DNA Bacteria protect their self DNA from restriction digestion by methylation of its recognition site. Methylation is adding a methyl group (CH 3 ) to DNA. Restriction enzymes are classified based on recognition sequence and methylation pattern.

5. Palindrome, Restriction Enzyme, Sticky Ends GAATTC G AATTC G Sticky Ends (Cohesive Ends) EcoRI CIVIC, Madam Get An Apple To The Class G AATTCG

6. Restriction Mapping of DNA A B 10 kb 8 kb 2 kb A 7 kb 3 kb B 5 kb 3 kb 2 kb A+BA+B CK A B A+B M Restriction enzymes Juang RH (2004) BCbasics

7. The Specific Cutting and Ligation of DNA GAATTC CTTAAG GAATTC CTTAAG G CTTAA AATTC G G G CTTAA G AATTC G G CTTAA AATTC G G CTTAA AATTC G EcoRI DNA Ligase EcoRI sticky end

8. CLONING

9. CLONING VECTORS

10. PLASMIDS Extra-chromosomal DNA found in bacteria. Loops of double-stranded DNA and some of them present in multiple copies. Independently replicate inside bacteria. Purpose of a vector is to carry DNA into a cell. Foreign DNA must be cut with same enzyme as plasmid so the ‘ends’ are compatible – plasmid and foreign DNA are then joined by ligase enzyme. Artificial plasmids have been genetically engineered for the purpose of cloning. Used as vectors, i.e., to transfer DNA from one cell to another.

11. Features of plasmids Plasmids also contain a selectable marker. Usually an antibiotic resistance gene: –required for maintenance of plasmid in the cell –advantageous for bacteria to keep the plasmid (can then grow in presence of antibiotic). Commonly used selectable markers are ampicillin, neomycin and chloramphenicol.

12. Cloning vectors In gene cloning, once recombinant DNA (rDNA) has been constructed it is introduced into a host. In the host, rDNA has to be: maintained replicated passed from one generation to another. This is achieved by introducing rDNA into a cell on a DNA vehicle called a cloning vector – most commonly used are plasmid cloning vectors.

13. Drug Resistance Gene Transferred by Plasmid Plasmid gets out and into the host cell Resistant Strain New Resistance Strain Non-resistant Strain Plasmid Enzyme Hydrolyzing Antibiotics Drug Resistant Gene mRNA

14. Target Genes Carried by Plasmid 1 plasmid 1 cell Recombinant Plasmid Transformation Target Gene Recombination Restriction Enzyme Restriction Enzyme Chromosomal DNA Target Genes DNA Recombination Transformation Host Cells

15. What is Bacterial Transformation? The transformation of bacteria! The genetic information of a bacterial cell actually takes in new genetic information and makes it a part of itself! It can then copy that sequence over and over and over and over and over ………………

16. Steps in transforming bacteria 1.Bacteria incubated with plasmid (with appropriate promoter and antibiotic resistance gene). 2.Bacteria plated out onto agar plates containing antibiotic. 3.Only those bacteria that have taken up plasmid will be able to grow on agar plus antibiotic. 4.Transformed bacteria can then be isolated and grown in bulk with appropriate antibiotic. 5.Bacteria multiply to produce genetically identical offspring – clones.

17. Amplification and Screening of Target Gene 1 1 cell line, 1 colony X100 X1,000 Plasmid Duplication Bacteria Duplication Plating Pick the colony containing target gene =100,000

18. Questions?

19. THANKS