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

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

DNA-Polymerase + Nucleotides

Primers

Denaturation 95°C

Annealing 50-60°C

Extension 72°C

Denaturation, annealing

Extension

x30

Steps in PCR

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/

• BLAST : http://www.ncbi.nlm.nih.gov/BLAST/

• Primer3 program:

http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

5' GATACCAGATGCATACGGCAACTAT 3'3' TATCAACGGCATACGTAGACCATAG 5'

5' CGAGATCATC

GATGATGCATCTAG 3'

A

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 enzymesNucleases:

• 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

T A

TA

TA

GC

G C

G C

GC

TA

T AGC

Restrictionenzymecuts here

AA

AA T

T

TA

G C

G CGC

GCGC

TT

Each strand has a ”sticky”end

Strandsseparate

A sticky end:

Restrictionenzymecuts here

T A

TA

TA

GC

G C

G C

GC

TA

T AGC

Restrictionenzymecuts here

Strandsseparate

TAGC

G C

GC

TA

T A

TAG C

T A

GC

Each strand has a ”blunt”endRestriction

enzymecuts 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