lecture 7- nucleic acid amplification

PCR

makes millions of copies of a specific DNA sequence so it can be detected and analyzed

denaturation

separation of the DNA strands in PCR

94 degrees celsius

temperature of denaturation in PCR

annealing

primers hybridize to separated strands in PCR

extension

synthesis of new strands by DNA polymerase in PCR

72 degrees celsius

what temperature should annealing happen at in extension of PCR

amplicon

replicated DNA

template DNA

essential for PCR; what we are copying

primers

essential for PCR; tells PCR where to copy

RNAse/DNAse free water

essential for PCR; need so we don’t break down what’s being copied

thermostable polymerase

essential for PCR; makes new DNA and needs to withstand high temps

dNTP mix

essential for PCR; DNA building blocks

MgCl2

essential for PCR; helps polymerase work

PCR buffer

essential for PCR; keeps reaction stable

taq polymerase

most commonly used thermostable polymerase

pfu polymerase

high fidelity thermostable polymerase; used when mistakes matter

primers

determine specificity of reaction

15-25 nucleotides long

how long are primers

40-60%

GC content in primers

4(GC)+2(AC)

equation for melting temperature

qPCR

also known as real time detection; measures DNA while PCR is happening using fluorescence

SYBR green

binds any double stranded DNA, cheap, and less specific; used in qPCR

taqman probe

binds only target sequence; more specific and more expensive; used in qPCR

conventional PCR

used gel electrophoresis to measure DNA

absolute quantification

using a standard curve of whole genomic DNA and plasmid with target sequence

relative quantification

quantity of gene in a sample is measured relative to that in another sample

hot start PCR

prevents non specific amplification during the set up of PCR reaction; needs high temperature to be activated

nested PCR

increases sensitivity and specificity; uses 2 rounds of PCR

multiplex PCR

used to detect multiple targets at once using probes with different colors

reverse transcriptase PCR

used for gene expression and RNA viruses

false positive

test says DNA present but it is not

false negative

DNA is present but test says its absent

contamination

cause of a false positive

negative control

what should be included to prevent a false positive

uracil DNA glycosylase

what can be used to prevent false positives that breaks anything with uracil in it

internal control

used to prevent false negatives; spikes sample with reference genes, if amplification doesn’t occur, something is wrong

positive control

what should be used to prevent false negatives