Polymerase Chain Reaction
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Transcript of Polymerase Chain Reaction
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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