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Polymerase Chain Reaction (PCR) is a revolutionary technique that enables the amplification of specific DNA sequences from minimal starting material such as blood, skin cells, or bone. By producing billions of copies of target DNA, PCR facilitates the isolation and analysis of DNA fragments. This process involves specialized ingredients including target DNA, primers, Taq polymerase, and dNTPs, and occurs in cycles of temperature changes (denaturation, annealing, and extension) in a thermal cycler. PCR plays a crucial role in genetics, forensic science, and medical diagnostics, advancing our understanding of human DNA, including genetic conditions like Huntington’s disease.
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Polymerase Chain Reaction PCR (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
What DNA is Used? • 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
What ingredients are needed? • 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
How Does PCR 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 What happens at each temperature change?
Denaturing Temperature • 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.
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 Annealing 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 Extension Temperature
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
D stands for the chromosome, and the S stands for map location of the chromosome, and the 80 is the locus point D1S80 • 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
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
