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

194 Cremer and Moos
5. Compare the value determined by qPCR with the result of the analysis by flow cytometry.
If the result obtained by qPCR is definitely lower than the one obtained by flow cytometry,
then the ASO primer is most probably not able to detect a single copy of the template
of the malignant clone.
3.7. Optimization of PCR Conditions for ASO Primers
1. As a starting point, use PCRs of 50 µL containing 5 µL of 10× PCR buffer, 2 mM MgCl 2 ,
each deoxynucleotide at 0.1 mM, the ASO primer pair or the CDR3-specific primer
plus LJH at 0.8 µM each, 2.5 U Taq-DNA-polymerase, and a maximum of 1000 ng
of DNA. Amplify with a program consisting of 7 min preheating at 94°C, 60 cycles of
1 min of denaturation at 94°C and 1 min of annealing and extension at 63°C, ending with
a final extension at 63°C for 5 min.
2. If these amplification conditions lead to unsatisfactory results, for example, too many
nonspecific products or low intensity of the specific product, optimize the reaction
conditions. Always amplify a negative control without added DNA, a negative control
with buffy coat DNA from healthy donors, and a positive control with DNA from a BM
sample of the patient.
3. Vary the annealing temperature in steps of 1°C. Vary the MgCl 2 concentration in steps of
0.25 mM. These parameters work synergistically.
4. Vary the concentration of primers in 0.2-µM steps in a range of 0.4 to 1.2 µM. Higher
concentrations of primers can lead to a higher sensitivity; however, this is hampered by
the occurrence of more nonspecific products.
5. Increase the number of amplification cycles to improve sensitivity. Decrease the number
of cycles to reduce nonspecific products. At least 50 cycles should be performed to allow
detection of single copies of template, which is a prerequisite for the statistical analysis of
limiting dilution series by likelihood maximization.
4. Notes
1. No distinct band or multiple bands are visible after consensus PCR: Some BM samples will
not lead to a single distinct band of the expected size after consensus PCR. Either only a
polyclonal smear is visible or, in rare cases, more than one band of the expected size. (1)
Change the strategy used for consensus PCR (FR3 or FR1). Try DNA instead of RNA.
Prepare a PCR mixture containing 10 µL of 10× PCR buffer, 2 mM MgCl 2 , each deoxynucleotide
at 0.05 mM, primers FR1C or FR3A plus LJH at 0.4 µM each, and 2.5 U Taq-
DNA-polymerase. Add 500 to 1000 ng of DNA and distilled water to a final volume of 100 µL.
Amplify with a program consisting of 7 min denaturation and enzyme activation at 94°C,
followed by 50 cycles of denaturation for 1 min at 94°C and combined annealing and
extension at 63°C (for primer FR3A) or 65°C (for primer FR1C), followed by a final
extension step at 65°C for 5 min. If possible, collect another BM aspirate and start again.
2. Sequence has no resemblance to known CDR regions: This is most probably caused by a
nonspecific product of the consensus PCR. Perform consensus RT-PCR again.
3. Sequence of a CDR-region is unsuitable for primer design: Sometimes the sequence of
a CDR region is too short, has too many base repeats, or has too many long stretches
rich in G and C, which makes designing primers difficult. Because CDR1 and CDR2
region are shorter and less variable than the CDR3-region, one should never do without a
CDR3-specific primer. If segments suitable for primer design are not long enough, identify
at least a short segment that is apt. Devise primers complementary to this stretch, and
elongate in 5′-end direction (never in 3′-direction, because this end is more important for
specific annealing of the primer to the template). Use the CDR1/2- plus CDR3-specific
primer strategy, if the CDR3 region is the one that is difficult for primer design.