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16 Cards in this Set
- Front
- Back
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PCR Concept |
Amplify single or few copies of piece of DNA -across several orders of magnitude |
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PCR components |
DNA + Deoxyribonucleotides + Taq + primers |
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Denaturation |
Heat 90-96 degrees for 20 seconds -Breaks DNA into single stranded molecules |
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Annealing |
Cooling 40-68 degrees for 20 seconds -Primers anneal to each end of segments -Taq polymerase synthesis complementary strands starting at primer |
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Extension |
Re-heat 70-75 degrees for 30 seconds -strands separate again -cycle continues |
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PCR Specificity |
Specific region can be targeted by primers -depends on position and amplicon length |
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PCR Sensitivity |
Takes only single copy of DNA to produce millions |
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Primer DImer |
Hybridization of primers to each other -They attach at 3' end and elongate -Compete for amplification with PCR reagents -DNA targeted site doe not amplify -Fix by changing enzyme |
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Misprimes |
Non-specific hybridization of primers -There is no specific amplification -primers had homologies |
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PCR Contamination |
Most commonly left over PCR product |
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PCR COntamination Prevention |
Air locks, positive airflor, hoods with UV -10% bleach, DNA AWAY |
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Multiplex PCR |
More than 1 pair of primers added to same rxn -Amplify multiple DNA simultaneously -Detects pathogens at same time -Decreases efficiency -Need to add internal positive to detect false neg |
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Nested PCR |
2 primer pair used to amplify target in 2 runs -2nd pair designed to bind inside of first -increases sensitivity of detection -used to low quantity DNA |
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RT PCR |
Revers Transcription PCR: two steps -Make cDNA -Run PCR -Measures RNA expression profiles -Detects micro organisms with RNA |
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Reverse Transcriptase |
Enzyme to make complementary DNA cDNA -From an RNA -Associated with retroviruses |
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Sequence-Specific PCR |
Requires complementarity of 3' primers -Used to identify base mutations -Point mutations show up when extension does not take place b/c lacking complementarity |
