L5: Nucleic Acid Amplificatoin

PCR

Primer-directed in vitro enzymatic reaction for amplification of a specific DNA fragment

fast with high-yield amplification

Foward Primer Purpose

defines 5’end of amplicon

Reverse Primer Purpose

defines 3’ end of amplicon

Three Steps of PCR

1. Denaturation of template

2. Annealing of primers

3. Extension of new strand

Annealing Temperature and Stringency

dependent on G+C content

May be high (no mismatch allowed) or low (allows some mismatch) stringency

Primers

ss DNA fragments complementary to sequences flanking the amplification region

distance between the primer binding sites determine PCR product size

Larger primer size = ?

greater annealing temperature

Primer Tm

52ºC-58ºC optimal

Tm of forward primer ≅ Tm of reverse primer (+/-2ºC)

Primer G/C content

40–60% with a 3’ position G or C if possible

Primers should avoid..

avoid primer complementarity

• Inter-strand complementarity (primer dimers)

• Intra-strand complementarity (secondary structure)

avoid repeats or runs

PCR Buffer

Buffering agent (Tris), Salt, and divalent cations

PCR Buffer: Salt Impact

affect the annealing and denaturing of the DNA (stringency) due to increase of Tm

monovalent

PCR Buffer: Divalent Cation Impact

(ie MgCl2) important for DNA polymerase activity

• too low = lower enzyme efficiency

  • too high = too many dNTPs misincorporated

Annealing Purpose

controls specificity of hybridization

Blank Rxn Ctrl and Reagents

controls for contamination

contains all reagents except DNA template

Negative Rxn Ctrl and Reagents

Controls for specificity of the amplification reaction

Contains all reagents and a DNA template lacking target sequence

Positive Rxn Ctrl and Reagents

Controls for sensitivity

Contains all reagents and a known target-containing DNA template

Amplification Rxn Ctrl and Reagents

distinguishes between true and false negative reaction

2nd set of primers and unrelated target

Extraction Ctrl and Reagents

confirm success of the extraction

control DNA sequence into lysis buffer with the target sample prior to DNA extraction.

control primers and probe for amplification

Misprime Reason

may occur due to non-specific hybridization of primers

Avoiding Misprimes

Use proper annealing temperature.

Design primers carefully.

Use hot-start

Hot-start

prepare reaction mixes on ice, place in preheated cycler or use an enzyme that requires an initial heat activation

Primer dimers Reason

may occur due to hybridization of primers to each other.

Avoiding Primer Dimers

Size is the sum of two primer lengths.

Taq extends one primer, which is annealed to another primer.

Annealing temperature too low = excess primers.

qPCR

Standard PCR with added probe/dye to generate a fluorescent signal from product, allows quantification of

starting material

With dilutions of known standards, you can determine the starting copy number by Ct

qPCR Taqman Probe

TaqMan recognizes target and extends DNA 5’-3’ exonuclease activity to chop up probe releasing reporter from quencher.

qPCR Plot

increase in fluorescence versus cycle number, grows exponentially

qPCR Plot: Baseline

initial PCR changes in which there is little change in fluorescence signal

qPCR Plot: Threshold

level of signal that reflects statistically significant increase over calculated baseline signal (typically 10x the SD of baseline signal

Ct (Threshold Cycle) Value

cycle number at which fluorescent signal of rxn crosses threshold

Digital > qPCR

extremely sensitive/ precise measurements

if target is present at low levels or mixed with a large amount of background DNA

partitions sample into thousands of tiny reactions and counts how many are positive, giving absolute number of target molecules without relying on standard curves

more accurate for detecting rare mutations, low-copy viral loads, or small foldchanges

What is the purpose of divalent cations ( Mg2+) in the PCR reaction?

Taq and other DNA polymerase enzymes require divalent cations for optimal activity.

Why must forward and reverse primer T_m be similar?

Must bind to their complementary sequences at the annealing step

Unequal primer binding will lead to poor PCR or no amplification.

From what do primer dimers result?

From 3′ complementarity in the forward and reverse primer sequences.

“self-priming” produces short products recognized as templates and are amplified

Why are dUTP and uracil glycosylase added to qPCR reactions?

control for contamination

dUTP places uracil in the PCR products in place of thymine, any contaminating product from this reaction will be digested by uracil glycosylase.

What type of probes covalently attaches fluorescent molecules to the PCR products?

Scorpion or primer probes are primers tailed at the 5′ end with fluorescent probes

amplicons will be attached covalently to the fluorescent probes