Polymerase Chain Reaction

(PCR)

PCR• PCR produces billions of copies of a

specific piece of DNA from trace amounts of starting material. (i.e. blood, skin cells, bone)

• Allows scientists to isolate pure quantities of specific DNA sequences

• 230 = over 1 billion copies of a specific DNA fragment; large enough quantity to be analyzed

• 46 Chromosomes code for 30,000 to 50,000 genes; only 5% of your DNA– Exons = DNA that is coded or expressed into

proteins• Noncoding DNA has more diversity; since

this DNA rarely leaves the DNA to head to ribosomes– Introns = DNA that is rarely expressed– Increased number of mutations

• Target DNA – the DNA that needs to be copied• Primers – short pieces of DNA that are designed

to attach to each end of the DNA fragment that will be replicated

• Taq polymerase – enzyme that reads the DNA– Comes from the bacteria Thermus aquaticus– Lives in the hot springs in Yellowstone; doesn’t fall

apart in high temperatures• dNTPs – 4 nucleotides with the 4 different bases

that are needed to replicate DNA• Buffer – gives the best environment for the

enzymes to work

• PCR machine is known as thermal cylcer• Machine changes to three different

temperatures during one cycle• Average number of cycles per run is 30 to

40

• Temperature at 94°C– The target DNA falls

apart– The H bonds

holding the nitrogen bases together break

– 2 individual strands of DNA are now present instead of a double helix.

Annealing Temperature• Temp between 56-65

– Primers attach to the ends of the Target DNA that needs to be copied

– Annealing = attachment of the primers

– Attach to complimentary bases of target DNA

Extension Temperature• Temperature at 72°C

– Provides best temp for Taq polymerase to begin reading the DNA

– Taq polymerase will synthesize a second strand of complimentary DNA

– Taq polymerase always read target DNA from 3’ to 5’ end

Repeat 30 times

• The three temperature changes represents one cycle– Denature– Anneal– Extend

• Repeat 30 times 230 = over 1 billion copies of the Target DNA

• Once DNA is amplified (copied), it is visible on a gel

PCR Animation

By the 4th Cycle = 32 Copies

D stands for the chromosome, and the S stands for map location of the chromosome, and the 80 is the locus point

• Locus is on chromosome 1• Intron – noncoding region of Chromosome 1• Each person has two copies of D1S80, one from

each parent• VNTR – Variable Number of Tandem Repeats• Consists of a repeating 16 base pattern (10 repeats

to >40 repeats)• Depending on how many repeating patterns

present, determines the size of your D1S80 locus

Homozygous or Heterozygous

Determining your genotype!

Locus with variability

D1S80

Huntington’s Chorea• Found on Chromosome 4• Noncoding region that actually causes

genetic disease• People with Huntington’s have a section on

chromosome 4 that has 35 or more of three base repeating pattern CAG (trinucleotide repeat)

• CAG normally codes for glutamine• Huntington’s patients will have a long line of

glutamine produced

Effects of Huntingtons

Dominant Autosomal Disease