Presentation on theme: "Basic methods in genetics PCR; Polymerase Chain Reaction Restriction enzyme digestions Gel electrophoresis."— Presentation transcript:
Basic methods in genetics PCR; Polymerase Chain Reaction Restriction enzyme digestions Gel electrophoresis
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
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!
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 Heat activation at 95°C for “hot start enzymes”. Final elongation step at 72°C
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
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/http://www.ensembl.org/ BLAST : http://www.ncbi.nlm.nih.gov/BLAST/http://www.ncbi.nlm.nih.gov/BLAST/ Primer3 program: http://www-genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi
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.
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
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
Restriction enzyme cuts here Each strand has a ”sticky” end Strands separate A sticky end: Restriction enzyme cuts here
Restriction enzyme cuts here Strands separate Each strand has a ”blunt” end Restriction enzyme cuts here A blunt end:
The designing of digestion reactions: Sequence of the target DNA must be known Webcutter 2.0: http://www.firstmarket.com/cutter/cut2.html
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
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