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15 Cards in this Set
- Front
- Back
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Denaturation step |
Heat to 94-95C dsDNA --> ssDNA GC rich DNA may need more time/higher temp all enzymatic reactions stop |
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Annealing step |
50-70C primers can bind to ssDNA |
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Extension Step |
Addition of nucleotide at 3' end 72C |
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Requirements |
DNA sample 2 oligonucleotide primers dNTPs Taq polymerase Buffer w/ MgCl2 |
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Template DNA |
Genomic cDNA cloned DNA |
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oligonucleotide primers |
custom made choice of the primer sequence is essential |
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what factors should you consider when designing primers? |
length base composition (avoid repetition, avoid base complementarity between 2 primers) melting temperature |
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Visualising PCR products via gel electrophoresis |
place in agarose gel containing ethidium bromide ethidium bromide intercalates DNA and fluoresces under UV electric current passed through the gel photograph taken for analysis |
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Advantages of PCR |
Fast and easy Sensitive Robust |
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Disadvantaged of PCR |
Requires template short PCR amplimer size infidelity - no proof reading |
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Variations of PCR |
Multiplex PCR - amplify several DNA samples in the same tube, requires various primers Produce labeled products - incorporate dNTPs that are labeled Amplify DNA of unknown sequence - lots of primers (degenerate) |
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Applications of PCR |
Clinical diagnosis Forensics Archeology Anthropology Food and agriculture industry - food testing |
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Clinical applications for PCR |
genotyping genetic markers - RFLPs, STRPs, tissue typing detection of mutations - cancer/genetic diseases Detection of pathogenic organisms in clinical samples |
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Forensic applications of PCR |
Genotyping (DNA fingerprinting) suspect or evidence matching ID human remains |
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Genetic diagnoses using PCR |
sickle cell thalassaemias CF huntingtons familial cancer syndromes muscular dystrophies |
