Recombinant DNA technology

1. Recombinant DNA technology

2. RECOMBINANT DNA TECHNOLOGY • RDA INVOLVES • ISOLATION + MANIPULATION OF DNA • TO MAKE CHIMARIC MOLECULES • CHIMARIC DNA/Recombinant DNA= HYBRID MOLECULE (molecule containing both HUMAN + BACTERIAL DNA SEQUENCES)

3. Manupulation of DNA – isolation and cloning Stages: 1. Generation of DNA fragments • By splicing by restriction endonucleases [RE]. • Splicing DNA fragment with sticky or blunt ends. 2. Separation of DNA fragments – by agarose gel electrophoresis/PAGE– Selection of desired piece of DNA 3. Insertion of selected DNA into cloning vectors- to create a Chimeric DNA 4. Introduction of these recombinant vectors into host cells e.g. bacteria 5. Manipulation and selection of clones containing recombinant molecules 6. Expression of genes to produce desired product.

4. Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.

5. 5’-GGATCC-3’ 3’-CCTAGG-5’ BamHI site: Restriction Enzyme Recognition Sites Restriction sites are general palindromic: “RATS LIVE ON NO EVIL STAR”

9. Chimeric DNA

13. The blot transfer procedure

14. Polymerase Chain reaction (PCR)

15. PCR- Polymerase Chain reaction • Test tube method of Amplification of a selected [target] DNA sequence • Permits synthesis of millions of copies of specific nucleotide sequences in a few hours-of very minute amount of sample • Sensitive, selective and extremely rapid • STEPS- • Primer construction • Denature DNA - 95 ˚C • Annealing of primers to ssDNA - 55 ˚C • Chain extension - 72 ˚C

16. Kary Mullis was awarded the 1993 Nobel Prize in chemistry for inventing PCR. • Temperature cycling – PCR process uses a machine (thermocycler) in which PCR reaction goes through ~30 cycles of three different temperature changes: ~95ºC – Melting temperature 50-65ºC – Annealing temperature 72ºC – Extension temperature

17. Polymerase chain reaction (PCR) analysis 1)Primers are designed to flank the region to be amplified in target DNA 2) Primers are annealed to denatured DNA 3) DNA is synthesized using Taq polymerase (from Thermus aquaticus) 4) Primers are annealed again and the process is repeated through 20-30 cycles, geometrically amplifying the target sequence 5) DNA is analyzed by gel electrophoresis

18. How PCR works: Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. Next, denature the DNA to single strands at 94˚C. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus. Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220)to 35 trillion copies (245) of the target DNA. Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. Cool to 4˚C and store or use amplified PCR product for analysis.

19. Hot water bacteria: Thermus aquaticus Taq DNA polymerase

20. PCR reaction process

23. Fig. 7.23 Denature Anneal PCR Primers Extend PCR Primers w/Taq Repeat…

25. Example thermal cycler protocol used in lab: Step 1 7 min at 94˚C Initial Denature Step 2 45 cycles of: 20 sec at 94˚C Denature 20 sec at 52˚C Anneal 1 min at 72˚C Extension Step 3 7 min at 72˚C Final Extension Step 4 Infinite hold at 4˚C Storage

26. Practical applications of PCR, Sequencing • Amplify DNA for Cloning (PCR) • Amplify DNA for sequencing without cloning (PCR) • Mapping genes and regulatory sequences • Diagnose disease • Pathogen screening • Sex determination • Forensic analysis • Paternity/maternity (relatedness) • Behavioral ecology studies (relatedness) • Molecular systematics and evolution (comparing homologous sequences in different organisms) • Population genetics (theoretical and applied) • Physiological genetics (studying basis of adaptation) • Livestock pedigrees (optimize breeding) • Wildlife management (stock identification/assessment)