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

190 Cremer and Moos
Fig. 3. Alignment of sequences of CDR3 consensus PCR products of the malignant clones
from 10 patients with MM and an example of four ASO primers designed for the last of the
given sequences (testing of the primers is shown in Fig. 4). Aligning allows to distinguish
variable and rather conserved segments. Primers are designed to be complementary to the
highly variable region.
3.4. Quantitative PCR (qPCR) Using ASO Primers
To quantitate PCR results, essentially four different methods have been described.
Although the measurement of the amount of PCR product, the coamplification of a
control gene, and the competitive PCR rely on the quantitation of the generated product,
the limiting dilution assay analyzes only positive and negative PCRs at different
dilution levels (13). Amplification efficiencies and hence the amounts of PCR product
can vary substantially from tube to tube despite identical and simultaneous processing
of reaction mixtures (14), even if quantitation is performed in the exponential phase of
amplification. Limiting dilutions are less prone to errors caused by these deviations.
Standards can be used to control for these differences; however, in MM a system of
standard and template would have to be established for each new patient. Therefore,
we prefer the limiting dilutions assay, which analyzes only PCR positivity versus
PCR negativity. A prerequisite of this method is that a single copy of target DNA
can be detected.
3.4.1. Designing Primers (see Note 3)
1. ASO primers should be 18 to 23 bases long.
2. The initiation of the Taq-DNA-polymerase can be enhanced by 3′-ends with NS or SS as
bases, but more than 2 G at the 3′-end should be avoided.
3. The 5′-end and 3′-end of primers should not be complementary to each other to avoid
primer homodimerization.
4. The A/T to G/C ratio should be approx 50%, if possible.
5. There are several computer programs that can be used to check primers for hairpin
formation and primer pairs for dimerization.
6. FR3-strategy: Identify hypervariable parts of the CDR3-region of the malignant clone
by aligning the sequence to known CDR3-sequences. Avoid segments that are relatively
conserved. Design ASO primer as forward primer according to above considerations. Use
ASO primer together with LJH as antisense primer.