Laboratory: Unit 3: PCR (pages 54-55)

Lecture: PCR & primer stock preparation

In-Class Writing: abstract for AEM 63: 2647-53, 1997 (page 68)

Hand In: abstract (page 68) Read: AEM 76: 8117-8125, 2010

Due Next Class: nothing

Factors that affect PCR:

Buffer: Mg2+, pH, dNTPs, salt, primer & template

Template quality & quantity

Facilitators (acetamide; 2-pyrolidone) Inhibitors (humic acid; hemoglobin)

Annealing temperature (primer length & G/C content; KCl concentration)

Initial temperature: hot start vs. cold start

Cycle number

Factors that affect PCR:

Primer sequence interactions with: itself, other primer, undesired templates

Polymerase fidelity & processivity

Cycler: temperature uniformity; ramp time; hot top

Reaction tubes: material & thickness

Ability of exclude or destroy extraneous DNAs

Uses of PCR:

Amplify DNA for sequencing; sequence amplicon (Ex. 3); clone & sequence (Ex. 4).

Add restriction sites to ends of a gene.

Detect small amounts of DNA.

Make specific mutations; overlap extension PCR.

Make "random" mutations; error-prone PCR.

Amplify unknown sequences flanking known sequence.

Uses of PCR:

Associate physical markers with genes: Random Amplification of Polymorphic DNA (RAPD).

cDNA cloning: Rapid Amplification of cDNA Ends (RACE).

Quantitative real-time PCR: dye fluoresces when bound to double-stranded DNA.

Abuses of PCR:

Use PCR to build a gene fusion but fail to sequence the clone; PCR is error prone.

PCR is extremely sensitive to DNA contamination. Quantitative measurements are more accurate using hybridization instead of PCR.

Aerosol resistant pipet tips are NOT aerosol proof.

Commercial dNTPs are contaminated with human and animal DNA. Other reagents may be contaminated too.

Abuses of PCR:

Inadequate no-template controls. Use 1 or 2 controls per sample.

Failure to determine sensitivity for each primer set & reaction cocktail.

Failure to remove PCR inhibitors. Dilution works well; requires no knowledge of inhibitor chemistry.

Abuses of PCR:

Cross contamination between samples when processing multiple samples due to inadequate cleanup of equipment & lack of physical precautions.

Use dedicated equipment, glove boxes, separate rooms for each step.

Use bleach to decontaminate equipment.

Abuses of PCR:

UV irradiate reagents to remove extraneous DNA. Ineffective if dNTPs present.UV destroys DNA polymerase if dNTPs absent.

Increase efforts to exclude extraneous DNA.

Reduce PCR cycles until contaminating DNAs not detected in no-template controls.

Dissolve 20-base primer (MW = 6600) in 1 ml water. Pipet 5 ul into 495 ul water. Absorbance @ 260 nm = 0.61 1 OD260 = 33 ug/ml for single-stranded DNA.

Concentration of undiluted primer stock?

MW = 6600 1M stock = 6600 g/l 1 uM stock = 6600 ug/l = 6.6 ug/ml

5 ul into 495 ul water = 1/100 dilutionOD260 of diluted stock = 0.61

concentrated stock = 0.61 OD260 x 100 x 33 ug/ml/ OD260

= 2013 ug/ml

2013 ug/ml x 1 uM/6.6 ug/ml = 305 uM

Dilute concentrated primer stock to 10 uM stock. You have 305 uM stock; you want 10 uM stock.

Divide what you want by what you have:

10 uM/305 uM = 0.0328 = 3.28/100

29 nmol/290 ul = 0.1 nmol/ul = 100 umol/l = 100 uM

25-nucleotide primer (50% G+C; 100% complementary to template) PCR contains 100 mM NaCl

What is melting temperature (Tm) of duplex DNA formed between primer and template DNA?

Tm = 16.6 log [Na] + 0.41 (% G+C) + 81.5 - 500/bp

[Na] = molar salt concentration % G+C = percentage (whole number; 50% = 50) bp = length of DNA:DNA hybrid in base pairs.

Tm = 16.6 log[0.1] + 0.41 x 50 + 81.5 – 500/25 = 16.6 (-1) + 20.5 + 81.5 – 20 = 65.5oC

Test of PCR Reagents

+ = template added; - = no-template control

bp

Fermentas100-bp

Gene RulerPlus

DNA ladder