PCR & DNA sequencing

human genetics lecture 6

polymerase chain reaction (PCR)

used to amplify small amounts of DNA and specific gene sequences

necessary components for PCR

• template DNA

• primers

• Taq polymerase

• deoxynucleotide triphosphate (dNTPs)

• magnesium ions

• buffer

• thermal cycler

Taq polymerase

enzyme that catalyze the adding of new nucleotides to the 3’ -OH of the primer (Thermus aquiticus)

deoxyribonucleotide triphosphate (dNTPs)

equal mixture of dATP, dCTP, dGTP, and dTTP

purpose of magnesium ions for PCR

required for Taq polymerase activity (co-factor)

thermal cycler

device that can change temperatures dramatically in a very short period

key steps of PCR

1. denaturation

2. annealing

3. elongation

denaturation

strands of DNA separate at 94°C, mimicking the action of helicase

annealing

the primers bind to the appropriate place on the template DNA at 40-65°C

elongation

• Taq polymerase synthesizes new DNA from 5’ to 3’ at 72°C

• DNA polymerase adds dNTPs to the primers

retrovirus

make a DNA copy of their genome and splice it permanently into the host’s cells

variable number of tandem repeat (VNTR) sequences

short, repeated sequences of nucleotides

Sanger method of DNA sequencing

fluorescently-tagged dideoxyribonucleotides (ddNTPs) are used to identify each base, when an altered nucleotide is added, synthesis stops and strands of every length accumulate

molecular phylogenetics

inference of evolutionary relationships based on molecular data