Basic methods in genetics

1. Basic methods in genetics • PCR; Polymerase Chain Reaction • Restriction enzyme digestions • Gel electrophoresis

2. PCR; Polymerase Chain Reaction • Amplification of specific DNA sequences • Invented by Kary Mullis in 1983 • Revolutionized the world of molecular biology • Mimics cell’s own DNA replication machinery

3. Ingredients in PCR: • DNA as a template • Thermo stable DNA polymerase enzyme • Deoxynucleoside triphosphates (dNTPs) • Synthetic oligonucleotide primers The flanking sequence of the target locus needs to be known!

4. Three major steps in PCR: • Denaturation: Strand separation at 95°C • Annealing: Hybridization of primers at 45-60°C • Extension: DNA synthesis at 72°C • Three steps are repeated for 25 to 40 times • Final elongation step at 72°C • Exponential increase of the number of copies → millions of copies of the target sequence

5. Primers DNA-Polymerase + Nucleotides Denaturation 95°C Annealing 50-60°C Extension 72°C Denaturation, annealing x30 Extension Steps in PCR

6. Technical problems • Contamination • Sensitivity to the levels of divalent cations • Quality of template DNA • Limited size of amplified product: • 300 bp-1000 bp are most efficient • possible to amplify fragments of several kb

7. Primer design • Must be very specific • No primer-primer interactions • No “hairpin” formation • No self-annealing • DNA sequence from the database (or from sequencing) • ENSEMBL http://www.ensembl.org/ • BLAST http://www.ncbi.nlm.nih.gov/BLAST/ • Primer3 program: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

10. What are enzymes? • Proteins that speed up chemical reactions in the body= protein catalyst essential to sustain life • Substrate = The molecule with which an enzyme interacts • Enzyme function is highly dependent on environmental characteristics such as temperature and pH.

11. Restriction enzymes Nucleases: • exonucleases: remove nucleotides from the end of DNA or RNA • endonucleases: make cuts at internal phosphodiester bonds Restriction endonucleases: • Discovered in the late 1960s • Found and purified from bacteria • Three types: • Type I and III: do not recognize a specific sequence to cut • Type II: cut specific recognized sequence

12. Type II enzymes: • Over 2500 different enzymes have been isolated • More than 500 enzymes are commercially available • Cut often sequence at palindromic hexanucleotide sequences • e.g. EcoRI : GAATTC • CTTAAG • Most enzymes cut within the recognition sequence • Leaves “sticky” or “blunt” ends

13. A sticky end: Each strand has a ”sticky” end Restriction enzyme cuts here Strands separate Restriction enzyme cuts here

14. Each strand has a ”blunt” end Restriction enzyme cuts here Strands separate A blunt end: Restriction enzyme cuts here

15. Restriction enzymes are powerful tools for molecular genetics Restriction enzymes can be used to: • Create restriction maps • Analyze sequence variations • Rearrange DNA molecules • Create mutants • Analyze the modification status of the DNA • ...other applications

16. The designing of digestion reactions: • Sequence of the target DNA must be known • Webcutter 2.0: http://www.firstmarket.com/cutter/cut2.html

19. Gel electrophoresis • A method that separates macromolecules on the basis of: • size • electric charge • other physical properties • Gel acts as a support medium • Electric field is generated across the gel • DNA is negatively charged → migration towards the positive pole • Small molecules move faster than big molecules • Ethidium bromide staining • Intercalates between bases of DNA • Can be visualized under UV-light

20. Requirements in gel electrophoresis: • An electrophoresis chamber and power supply • Gel casting tray • Sample comb • Agarose • Electrophoresis buffer • Loading buffer • DNA ladder (= size standard) • Ethidium bromide • Transilluminator