Molbio: Components of PCR

Primers

single-stranded DNA fragments (20-30 bases) designed to flank the target region

Primers

with forward and reverse primers binding to opposite strands.

Primer optimization

annealing temperature

Primer sequence and length

Primer optimization depends on?

dictate the size of the amplified product.

The placement of the primers will?

Primer Dimers

Double-sized PCR products formed by primer binding and copying, resulting from short homologies at the 3′ ends.

Primer Tm

can serve as a starting point for setting the optimal annealing temperature

Amount of dna template

Nanogram amounts (100 ng to 1 g)

contaminating proteins, nicks, or breaks, and have minimal GC content and secondary structure

Best templates are free of

Source of dna template

patient’s genomic or mitochondrial DNA, viruses, bacteria, fungi, or parasites.

Building Blocks of DNA

Nucleotide triphosphates, specifically deoxynucleotide-triphosphates (dNTPs).

Adenine

deoxyadenosinetriphosphate (dATP)

Guanine

deoxyguanosinetriphosphate (dGTP)

Thymine

deoxythymidinetriphosphate (dTTP)

Cytosine

deoxycytidinetriphosphate (dCTP)

higher than the estimated Km (10–15 mM) for optimal amplification.

dNTP Concentration

Taq polymerase

isolated from Thermus aquaticus

Tth polymerase

isolated from Thermus thermophilus

Proofing enzyme

allows DNA polymerases to generate large products of 30,000 bases in length

Modified Polymerases

Cloning of polymerase genes has led to the Stoffel fragment, lacking the N terminal 289 amino acids of Taq polymerase.

Stoffel Fragment

Has a longer half-life at high temperatures

High-Fidelity Polymerases

Modified Taq enzymes retaining 3′ to 5′ exonuclease activity but not 5′ to 3′ exonuclease activity are used.

20-100mM

Buffer Composition: Potassium chloride

15-30 mM

Buffer Composition: Ammonium sulfate

1-4 mM

Buffer Composition: Magnesium chloride

10mM

Buffer Composition: Tris-HCl

8-9.5

pH level of proper buffering for Tris-HCl

Master Mix Preparation

Buffer, nucleotide bases, and other ingredients are mixed and stored as aliquots for convenience.

Master Mix Components

Enzyme, target DNA, and primers are added to the master mix when needed

Thermal cyclers or thermocyclers

designed to rapidly and automatically ramp (change) through the required incubation temperatures, holding at each one for designated periods.

Rapid PCR System

Designed for small sample volumes with quick heating and cooling.

Real-time PCR System

Equipped with fluorescent detectors to measure PCR product during the reaction.

Microchip PCR

Performed in a microchip with tiny channels for sample flow and temperature control.

1–2 uL

Sample used in Microchip PCR

Positive control

ensures that the enzyme is active, buffer is optimal, and primers are priming right sequences

Negative control without DNA

ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run

Negative control with DNA

ensures that the primers are not annealing to unintended sequences of DNA.