Quantitative Analysis of the PCR Reaction

PCR Conditions for Lambda Control:M

  • 200 µM (each) dNTPs; total [dNTPs] = 0.8 mM
  • Total [MgCl2] = 1.5 mM
  • Free [MgCl2] = 0.7 mM
  • 2.5 Units AmpliTaq® DNA Polymerase per 100 µL
  • 1 micromolar (each) primers [PCR01, PCR02]
  • Bacteriophage Lambda DNA template
    (Template ds DNA = 48,500 bp)
  • Target = 500 bp
  • Number of cycles = 25
  • Buffer: 10 mM Tris-HCl, pH 8.3 (at 25 °C); 50 mM KCl

Before PCR After PCR

Weight Moles Molarity Molecules Weight Moles Molarity Molecules
Template (48,500 bp) 1 ng 3.10x10-17 3.10x10-13 1.86x107 1 ng 3.00x10-17 3.00x10-13 1.81x107
Target
(500 bp)
10 pg 3.00x10-17 3.00x10-13 1.81x107 1 µg 3.00x10-12 3.00x10-8 1.81x1012
Primers (25-mers) 1623 ng 2.00x10-10 2.00x10-6 1.20x1014 1574 ng 1.94x10-10 1.94x10-6 1.17x1014
dNTPs 39 µg 8.00x10-8 8.00x10-4 4.82x1016 37 µg 7.70x10-8 7.70x10-4 4.64x1016
Magnesium Ion 3.6 µg 1.50x10-7 1.50x10-3 9.03x1016 3.6 µg 1.50x10-7 1.50x10-3 9.03x1016
AmpliTaq® DNA Polymerase 12.5 ng 1.33x10-13 1.33x10-9 8.01x1010 12.5 ng 1.33x10-13 1.33x10-9 8.01x1010

Assumptions and Data:

  • AmpliTaq® DNA Polymerase specific activity
    = 250,000 Units/mg
  • Average MW of a dNTP is 487 Daltons
  • Average MW of a dNMP is 325 Daltons
  • Achieve at least 105-fold amplification
  • AmpliTaq® DNA Polymerase half-life is not considered

Spectrophotometric Conversions:

Double-stranded DNA (ds DNA):
A260 = OD260 = 1 for a 50 µg/mL solution

Single-stranded DNA (ss DNA):
A260 = OD260 = 1 for a 33 µg/mL solution

RNA: A260 = OD260 = 1 for a 40 µg/mL solution

Reference: Freifelder, D., Physical Biochemistry: Applications to Biochemistry & Molecular Biology, W.H. Freeman and Company, CA, 1982, p. 494-536.

Useful Equations and Nucleic Acid Molecular Weight Data:

Absorbance =
Molar Extinction Coefficient x Concentration x Pathlength

500 bp of double-stranded DNA = 325,000 Daltons
500 nt* of single-stranded DNA = 162,500 Daltons
Average MW of dNMP is 325 Daltons
(*nt = nucleotide)

Oligomer Quantitation:

For a 20-mer, a stock solution with A260 = 1 contains 5 nmol
5 nmol = 33 µg/(20 x 325)

For a 40-mer, a stock solution with A260 = 1 contains 2.5 nmol
2.5 nmol = 33 µg/(40 x 325)

Conversion of pmoles of primer to µg of primer:

Multiply pmoles by (length x 325)/1,000,000

Example: 10 pmoles of a 25-mer
(10 x 25 x 325)/1,000,000 = 0.081 µg primer

Conversion of µg of primer to pmoles of primer:

Multiply by 1,000,000/(length x 325)

Example:
0.1 µg of a 20-mer
(0.1 x 1,000,000)/(20 x 325) = 15.4 pmoles primer

Calculating Primer Concentrations for PCR Amplification:

Micromolar concentration of primer = pmoles/µL

Example 1:
20 pmoles of primer in 100 µL PCR mixture =
0.20 micromolar

Example 2:
Primer is 24 nucleotides in length and is dissolved in 0.1 mL
of water

A 10 µL aliquot is diluted to 1.0 mL for A260 measurement:
A260 = OD260 = 0.76

The stock solution has an absorbance at 260 nm (A260) of 76

The stock solution (0.1 mL) contains 7.6 A260 units

The base composition of the primer is A=6, C=6, G=6, T=6

The Molar Extinction Coefficient at 260 nm for the primer = a(16,000) + b(12,000) + c(7,000) + d(9,600)
where: a is the number of A's, b is the number of G's, c is the number of C's, d is the number of T's

The Molar Extinction Coefficient of the PCR primer is:
6(16,000) + 6(12,000) + 6(7,000) + 6(9,600) = 267,600

The Molar Concentration of the PCR primer stock solution is:
76/267,600 = 284 micromolar

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