John M. S. Bartlett.pdf - Bio-Nica.info

qPCR to Detect RNA Viruses 201
the samples can then be extrapolated by finding the detection end point. A specific
volume of cDNA may also be added to a number of replicate reactions to give a Poisson
distribution (5,14,15).
3.2.3. The Use of External Standard Curves
A 10-fold dilution series covering from 1 to 10 5 molecules is used to produce a
standard calibration curve. Amplified products can then be analyzed using the desired
detection system, for example, by microwell capture so that optical density values can
be compared with those of the standard curve (4).
3.3. Competitive PCR Using an Internal Control
3.3.1. Creation of a Mutated Template Containing a Novel RE Site
1. Perform two PCR reactions, each containing an external primer plus the appropriate
internal primer with mismatches to create the RE site. Approximately 20 to 30 cycles using
a low annealing temperature will allow for mismatches (Fig. 1).
2. Analyze the products by agarose gel electrophoresis, excise the bands, and recover the
DNA using Geneclean following manufacturer’s protocols.
3. Heat the combined products to 94°C for 1 min, then cool and allow to renature.
4. Amplify the renatured products using the external primers and normal cycling conditions.
5. Check for the mutated site by RE digest (3 µL of product, 1 µL of 10× buffer, 5 µL of
dH2 2 O, 10 U/µL enzyme) at the appropriate temperature for 1 h. Analyze products by
agarose gel electrophoresis. Alternatively, sequence the amplicon.
6. If required, the products may be cloned into a PCR cloning vector for further manipulation
or simply used as PCR template to create as much competitor as necessary.
3.3.2. Quantitative PCR Using the Mutated Competitor
1. Perform a series of 10-fold titrations of mutated fragment with known copy number
(1–10,000 copies; see Note 6).
2. Add each to a separate basic PCR along with the unknown quantity of DNA to be amplified
and perform as few rounds as necessary (20–30 rounds).
3. Run products on 1 to 2% agarose and analyze by densitometry (gel or photograph; see
Fig. 2).
4. The point at which wild-type and mutant intensity is equal identifies the amount of wild
type DNA to within 1 log.
4. Notes
1. The RT and PCR stages may be performed in a single-tube format and also combined with
hot start when wax beads are used to separate the stages (7).
2. PCR DIG labeling mix must also be included if using EIA detection methods. Instead
of adding combined dNTPs, add 200 µM of dATP, dCTP, dGTP, 190 µM of dTTP, and
10 µM of Dig dUTP.
3. Pfu has been found to generate higher product yields (4).
4. cDNA produced in this reaction appears to be slightly unstable. Long-term storage leads
to significant reductions in the amount of target DNA.
5. Hot start PCR where a heating step at 95°C is required to activate the polymerase may
increase amplification efficiency.
6. Further titrations can be performed, although this is expensive and time consuming.
7. The 118-bp fragment must also be taken into account when finding the point of equilibrium.
Although it is possible to detect the end point of this reaction by eye (between lanes 6 and 7),
it is more accurate to use densitometry.