Polymerase Chain Reaction

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Polymerase Chain Reaction (PCR)

description

Polymerase Chain Reaction. PCR. (PCR). P C R. 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 - PowerPoint PPT Presentation

Transcript of Polymerase Chain Reaction

Page 1: Polymerase Chain Reaction

Polymerase Chain Reaction

(PCR)

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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

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• 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

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• 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

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• 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

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• 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.

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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

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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

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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

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PCR Animation

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By the 4th Cycle = 32 Copies

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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

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Homozygous or Heterozygous

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Determining your genotype!

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Locus with variability

D1S80

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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

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Effects of Huntingtons

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Dominant Autosomal Disease