Endpoint Pcr For Dna Quantification

A less elegant (and less expensive) approach for testing the 'amplifiability' of a DNA sample is to perform an end-point PCR test. In this approach a single STR locus (Kihlgren et al. 1998, Fox et al. 2003) or other region of the human genome, such as an Alu repeat (Sifis et al. 2002, Nicklas and Buel 2003a), is amplified along with DNA samples of known concentrations. A standard curve

Kit or Assay

Principle Behind Detection

Limit of Detection

Volume of DNA Used

Human/Primate Specific

PCR Inhibition Detected

Reference

Quantiblot Kit

Hybridization

~150 pg

5 |L

Y

N

Walsh et al. (1992)

PicoGreen Assay

Intercalating Dye Fluorescence

250 pg

10 |L

N

N

Hopwood et al. (1997)

AluQuant Kit

Pyrophosphorylation and luciferase light production

100 pg

1-10 |L

Y

N

Mandrekar et al. (2001)

BodeQuant

End-point PCR

~100 pg

1-10 |L

Y

Y

Fox et al. (2003)

TQS-TH01

End-point PCR

~100 pg

1-10 |L

Y

Y

Nicklas and Buel (2003a)

Quantifiler Kit

Real-time PCR

20 pg

2 |L

Y

Y

Applied Biosystems (2003)

Alu Assay

Real-time PCR

1 pg

1-10 |L

Y

Y

Nicklas and Buel (2003c)

CFS TH01 Assay

Real-time PCR

20 pg

1-10 |L

Y

Y

Richard et al. (2003)

RB1 and mtDNA multiplex

Real-time PCR

20 pg

1-10 |L

Y

Y

Andreasson et al. (2002)

can be generated from the samples with known amounts to which samples of unknown concentration are compared. A fluorescent intercalating dye such as SYBR® Green (see Chapter 13) can be used to detect the generated PCR products. Based on the signal intensities resulting from amplification of the single STR marker or Alu repeat region, the level of DNA can be adjusted prior to amplifying the multiplex set of DNA markers in order to obtain the optimal results (see Chapters 5 and 6). This method is a functional test because it also monitors the level of PCR inhibitors present in the sample (see Chapter 7). In the end, each of the DNA quantitation methods described here has advantages and disadvantages and could be used depending on the equipment available and the needs of the laboratory.

Several inter-laboratory tests to evaluate DNA quantification methods have been conducted by the U.S. National Institute of Standards and Technology (NIST) to better understand the measurement variability seen with various techniques (Duewer et al. 2001, Kline et al. 2003). A ten-fold range of reported concentrations was observed in one study (Figure 3.4).

Most DNA quantitation measurements are precise to within a factor of two if performed properly (Kline et al. 2003). While this degree of imprecision may not seem reliable enough, quantitation results are usually sufficiently valid to estimate DNA template amounts that will enable PCR amplification.

1 ng sample

Q CO

-10 12 Concordance, SD

-10 12 Concordance, SD

Table 3.3 (Facing) Summary of various DNA quantitation methods. Commercially available kits are listed in bold.

Figure 3.4

(a) Range of DNA concentrations reported for a 1 ng DNA sample supplied to 74 laboratories in an inter-laboratory study (Kline et al. 2003). Overall the median value was very close to the expected 1 ng level with 50% falling in the boxed region. However, laboratories returned values ranging from 0.1-3 ng.

(b) A target plot examining concordance and apparent precision for the various laboratory methods used. Legend: A = ACES kit;

q = Quantiblot with unreported visualization method; E = Quantiblot with chemiluminescent detection; T = QQuantiblot with colorimetric detection; 1, 2, 3, 4, and 5 represent methods used by only one laboratory.

Calculation of DNA quantities in genomic DNA

Important values for calculations:

1 bp = 618 g/mol A: 313 g/mol; T: 304 g/mol; AT base pairs = 617 g/mol G: 329 g/mol; C: 289 g/mol; GC base pairs = 618 g/mol

1 genome copy = ~3 x 109 bp = 23 chromosomes (one member of each pair)

1 mole = 6.02 x 1023 molecules

Standard DNA typing protocols with PCR amplification of STR markers typically ask for 1 ng of DNA template. How many actual copies of each STR locus exist in 1 ng?

1 genome copy = (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol

= (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)

Since a diploid human cell contains two copies of each chromosome, then each diploid human cell contains ~6 pg genomic DNA

Therefore 1 ng genomic DNA (1000 pg) = ~333 copies of each locus

(2 per 167 diploid genomes)

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