2.4.1 General considerations
The major use of this type of assay to date is to monitor viral replication. Quantitative PCR technology is a powerful tool providing information into the pathogenesis of viruses by determining the viral load at a particular time point, or more informatively, by performing longitudinal analysis. Methods for quantitation are based on the comparison of two amplified signals one of which is of known quantity. The main objectives are to develop a fully quantitative assay over a broad dynamic range to allow quantitation of low
and high copy numbers of DNA. The two sequences for comparison in the quantitative assay i.e the internal control and target sequences, should be as closely related to each other as possible. In addition the control sequence should be added to each PCR reaction to rule out the possibility of tube to tube variation.
Several strategies for differentiation between the control and unknown target sequence have been employed including size differentiation by insertion or deletion of the internal control DNA, amplification with different primers of endogenous sequences that are functionally related , or the introduction of a unique restriction site into the control sequence itself. The latter of these strategies presents the best scenario since the difference between the target and the intemal control sequence is limited to a few bases, and can be achieved by simple mutagenesis. The co-amplification of an intemal control sequence with a unique restriction site together with the target DNA is the method of choice for this thesis. In order to determine the limits of a quantitative assay, the dynamic range must first be determined by production of a calibration curve, whereby the control and the target sequences are compared. Consideration of dynamic range is based on the fact that the PCR product itself accumulates exponentially up to the plateau phase of the reaction.
The quantitative assay used in this thesis is based on the co-amplification of a known copy number of intemal control with the target DNA present in the clinical sample which has been described previously in detail (Fox e ta l, 1992). The intemal control harbours a unique restriction site for the enzyme Hpa1 by mutagenesis of the wild type sequence
from the original GG to TT within the gB gene of HCMV. This mutant sequence was then sub-cloned into a pUC13 vector (Pharmacia) and purified as a source of intemal control DNA. The primers for the PCR are identical to those used for the qualitative assay described above with the exception that g82 is 5' OH labelled with [y-^^P]ATP to allow detection following the PCR reaction. Quantitation is achieved following digestion of the PCR product with Hpa^ which cleaves only the intemal control sequence into 77 and 72bp fragments leaving the target DNA in the sample of interest undigested. The digestion products are separated by polyacrylamide gel electrophoresis (PAGE) and subjected to autoradiography and scanning densitometry . Since a known quantity of intemal control is included in each PCR reaction, a direct comparison of the intensity of the target DNA with the cleaved intemal control allows the copy number determination of unknown target to be calculated. To ensure accuracy in this calculation, three tubes containing different known quantities of control sequence but the same amount of the
unknown target present in the clinical sample are assayed in parallel.
2.4.2 Phosphorylation of primer gB2
Initially to allow detection of the PCR products, one of the primers was 5' OH end labelled with [Y-^^P]ATP using T4 polynucleotide kinase (PNK). This enzyme catalyses the end labelling of 5' termini of DNA or RNA by transferring the y-phosphate of ATP to the 5' OH group of the primer. The gB2 primer was 5' phosphorylated by adding the reaction components in the order stated below at 37°C for 30min, the reaction was stopped by placing it at -70°C until use.
Labellina Reaction Forward Buffer
2^Â gB2 (250ngVI) 350mM Tris-HCL
5X Forward Buffer (Gibco BRL) 50 mM MgCl2
V I PN K (IO uVI) (Gibco BRL) 500mM KCL
1 0 ^ ( 1 [Y-^^P]ATP(100/.^Ci) (Amersham) 5mM 2-mercaptoethanol
6 6 / ^ 1 S D W
^00^\ Total
Once the primer had been labelled, the PCR reaction and cycling conditions were performed as for the qualitative PCR in section 2.3 with the exception that V I (5ngVI) of labelled gB2 primer was added. The target DNA in this case consisted of known copy numbers of both the intemal control and wild type sequence, both sequences were subsequently co-amplified in the same tube. Each tube contained the same copy number of the intemal control, with the following range for the wild type sequence; 1 0®, 1 0\ 1 0^, 1 0^ and 1 0 copies.
Quantitative PCR reaction mixture for one tube: 10/il 10X GeneAmp 10X PCR buffer
8;/l MgCLj (25mM)
Z^A dNTP mixture (200/.iM each) (Promega) V I gB I (lOOng)
V I gB2 (lOOng)
V I gB2 * ([Y-""P]ATP 5ng/ul)
0.25/.il AmpliTaq Gold (5u V I) (Amplitaq gold) 71.75 //I S D W
(2//I Intemal control DNA sample) (2//I Wild type control DNA) 100//I Total
The reaction mixture was overlayed with 100/^1 of molecular biology grade mineral oil (Sigma).
Note: As with the qualitative PCR, the intemal and wild type sequences were added under the oil in a separate PCR set up area as were the negative w ater controls added every fifth tube to assess for contamination problems.