Basic methods in geneticsPCR; Polymerase Chain ReactionRestriction enzyme digestionsGel electrophoresis

PCR; Polymerase Chain Reaction

Amplification of specific DNA sequencesInvented by Kary Mullis in 1983Revolutionized the world of molecular biologyMimics cells own DNA replication machinery

Ingredients in PCR:DNA as a templateThermo stable DNA polymerase enzymeDeoxynucleoside 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 95CAnnealing: Hybridization of primers at 45-60CExtension: DNA synthesis at 72C

Three steps are repeated for 25 to 40 timesFinal elongation step at 72CExponential increase of the number of copies millions of copies of the target sequence

Steps in PCR

Technical problems

ContaminationSensitivity to the levels of divalent cationsQuality of template DNALimited size of amplified product:300 bp-1000 bp are most efficientpossible to amplify fragments of several kb

Primer designMust be very specificNo primer-primer interactionsNo hairpin formationNo self-annealing

DNA sequence from the database (or from sequencing)ENSEMBLhttp://www.ensembl.org/BLASThttp://www.ncbi.nlm.nih.gov/BLAST/

Primer3 program: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

What are enzymes?

Proteins that speed up chemical reactions in the body= protein catalyst essential to sustain lifeSubstrate = The molecule with which an enzyme interactsEnzyme function is highly dependent on environmental characteristics such as temperature and pH.

Restriction enzymes

Nucleases:exonucleases: remove nucleotides from the end of DNA or RNAendonucleases: make cuts at internal phosphodiester bonds

Restriction endonucleases:Discovered in the late 1960sFound and purified from bacteriaThree types:Type I and III: do not recognize a specific sequence to cutType II: cut specific recognized sequence

Type II enzymes:

Over 2500 different enzymes have been isolatedMore than 500 enzymes are commercially availableCut often sequence at palindromic hexanucleotide sequencese.g. EcoRI : GAATTC CTTAAGMost enzymes cut within the recognition sequence Leaves sticky or blunt ends

A sticky end:Restrictionenzymecuts here

Restrictionenzymecuts here A blunt end:

Restriction enzymes are powerful tools for molecular geneticsRestriction enzymes can be used to:Create restriction mapsAnalyze sequence variations Rearrange DNA moleculesCreate mutantsAnalyze the modification status of the DNA...other applications

The designing of digestion reactions:

Sequence of the target DNA must be knownWebcutter 2.0: http://www.firstmarket.com/cutter/cut2.html

Gel electrophoresisA method that separates macromolecules on the basis of:sizeelectric chargeother physical propertiesGel acts as a support mediumElectric field is generated across the gelDNA is negatively charged migration towards the positive poleSmall molecules move faster than big moleculesEthidium bromide stainingIntercalates between bases of DNACan be visualized under UV-light

Requirements in gel electrophoresis:An electrophoresis chamber and power supplyGel casting traySample combAgaroseElectrophoresis bufferLoading bufferDNA ladder (= size standard)Ethidium bromideTransilluminator