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

Site-Directed Mutation and PCR Mimics 209
for samples assayed within a single assay the variation can be kept within acceptable
limits. Furthermore, although it is possible, using the pCRII vector, to generate mRNA
as an additional control, the low intra assay variation defined in this system allows this
simpler procedure to be followed.
Interassay and intra-assay variations were similar to those obtained by conventional
radioimmunoassays (2,3), suggesting this technique could be robustly applied to
clinical diagnostic problems, such as the determination of viral load for either RNA or
DNA viruses (omitting the RT step for the latter).
The sensitivity of this technique is such that low copy number genes could be
assayed in 100 s or at most a few thousand cells and can be applied to patient tissue
samples and small cell cultures. In addition, by allowing absolute concentrations to
be determined, this assay will facilitate comparisons between laboratories previously
hampered by semiquantitative approaches to PCR (4,5), and this precision is maintained
even over most applications of fluorescence real-time PCR (6).
4. Notes
1. The cDNA sequence for human cAMP-dependent protein kinase subunit was retrieved
from the Gembl database (accession no. M33336; 7). Using this sequence, primers were
designed that amplified bases 159 to 589. This 430-bp fragment codes entirely for mRNA,
which is subsequently translated into protein.
2. The mimic is inserted in the 3900-bp pCRII vector by calculating the length of the vector
containing the insert and dividing this by the length of the insert the molecular weight fraction
made up by the insert is calculated and the Mwt corrected to reflect that of the PCR insert
only. The molar equivalent for RI alpha mRNA added to the assay system was calculated
as follows: 1 pg RI alpha plasmid is equivalent to 0.34 fmols RI alpha mRNA.
3. The PCR mimic was constructed as described elsewhere. Using site-directed mutagenesis,
a single bp change was introduced within the RI alpha PCR product to introduce a EcoRV
restriction site. For researchers wishing to evaluate this quantitative PCR approach, this
mimic is available on request from the author.
4. Reverse transcription with a random hexamer will target all RNA species. Specific primers
for the product of interest, out with the PCR product sequence, or polyA primers to target
mRNA may be substituted as required.
5. Because of the high activity of 32 P, alternatives such as 33 P or biotin-dCTP may be
considered.
6. Magnesium chloride concentrations may need to be altered for different PCR products. We
therefore recommend using buffer with separate magnesium chloride.
7. Using heated lids avoids the need for paraffin oil overlay, which reduces evaporation.
8. To evaluate the reproducibility of the reverse transcription reaction we recommend
including 0.5 µCi [ 35 S]dATP. This small amount of [ 35 S]dATP does not affect subsequent
quantitation of the PCR. In our previous experiments, we demonstrated an intra-assay
variation of 17% between samples in RT reactions and a 9% mean inter assay variation.
9. The sensitivity of the assay can be varied by varying the standard range; decreasing the
range of mutant and control plasmid from 0.1 to 100 pg to 0.01 pg to 1.0 pg increased the
sensitivity of detection. For these experiments, PCR was performed over 30 cycles.
10. The restriction enzyme EcoRV has an optimal digestion in buffered 50 mM KCl, 10 mM
NaCl, and addition of 10% 100 mM NaCl to the PCR mix best approximates these
conditions and avoids the need for purification of the PCR product at this step. The use of
amplified control and mutant plasmids provides a high degree of control over the efficiency
of the restriction digestion reaction.