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

192 Cremer and Moos
Fig. 4. Testing of primers (see Fig. 3) as described in Subheading 3.4.2. For each ASO
primer, a negative control without added DNA, a positive control with DNA from the initial BM
sample, and a specificity test with buffy coat DNA were amplified by PCR. Using primer 1, only
the positive control leads to the PCR product of the expected size. Primers 2, 3, and 4 produce
several nonspecific bands, even in the negative control. Primer 3 generates a false-positive PCR
product of the same size with buffy coat DNA. Primer 1 was selected, and PCR conditions were
further optimized for this oligonucleotide as described in Subheading 3.7.
undiluted level has been reached) to partial PCR positivity in the next higher dilution levels
to PCR negativity in all replicates in the highest dilution levels (see Fig. 5).
9. To deduce the proportion of malignant cells in the undiluted sample from the pattern
of PCR results, analyze by likelihood maximization and by χ 2 -minimization (15), for
example, by using the MAXLIKE computer program. Ten microliters of the undiluted
DNA solution (100 ng/µL) are equivalent to 165,000 cells, which is used as a starting
point for the calculations. The program will return the most probable value for the initial
proportion of malignant cells in the analyzed sample.
10. Compare the values obtained by likelihood maximization and by χ 2 -minimization. If PCR
results are plausible, both methods will yield consistent values (see Note 5).
3.6. Testing the Sensitivity of ASO Primers
A prerequisite for the analysis of the pattern of PCR results by χ 2 -minimization
or likelihood maximization is that a single copy of target DNA per PCR tube can be
detected. It is advisable that at least the overall sensitivity of the assay should be tested,
as described in Subheading 3.6.1.