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

PCR Detection of Tumor Cells 191
7. FR1-strategy: Distinguish FR regions and CDR regions by aligning sequence to known
CDR sequences. Design the first ASO primer as the forward primer complementary to the
CDR1 or CDR2 region (CDR1 regions are often rather conserved, thus CDR2-specific
primers might be preferred). Design the second ASO primer as the antisense primer
complementary to the CDR3 region. Use these primers as a pair for PCR.
3.4.2. Testing of ASO Primers
Because even carefully designed primers may not work as well as expected, only
testing can distinguish between oligonucleotides that fulfill all requirements and
those that are not specific or sensitive enough. CDR1/2- plus CDR3-specific ASO
primer pairs do not necessarily give better results than CDR3-specific plus J-consensus
primers. An example of a primer test is given in Fig. 4. Design several CDR3-specific
or CDR1/CDR2-specific plus CDR3-specific (antisense) primers. An example for 4
ASO primers is given in Fig. 3.
1. Prepare PCRs 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-CL at 0.8 µM each,
and 2.5 U Taq-DNA-polymerase.
2. Add 500 ng of DNA from the initial BM sample to generate a positive control. Add 1000 ng
of buffy coat DNA from healthy donors to generate a negative control that tests for
specificity of the primers. Adjust with distilled water to a final volume of 50 µL. Repeat
this for every primer combination to be tested.
3. Amplify with a program consisting of 7 min of preheating at 94°C, 60 cycles of 1 min
of denaturation at 94°C, and 1 minute of annealing and extension at 63°C, ending with
a final extension at 63°C for 5 min.
4. Analyze PCR products on a 2 (for FR1/2-strategy) or 5% (for FR3-strategy) ethidium
bromide-stained agarose gel. Reactions containing DNA from the positive control should
display a prominent band of the expected size (see Note 4). No such product should be
visible in those with buffy coat DNA only. Avoid primers that generate a multitude of
nonspecific products.
3.5. Quantitation by Limiting Dilutions
1. Isolate DNA from the sample to be assessed (see Note 3).
2. Determine the DNA concentration of the sample by OD measurement. Add distilled water
to achieve a DNA concentration of 100 ng/µL.
3. Prepare a dilution series in 0.5 log steps (resulting in dilution levels of 13, 110,
133, etc.).
4. For each dilution level, set up a PCR containing 10 µL of the DNA solution. Use conditions
as determined to be optimal for the ASO primers used (see Subheading 3.7.) and
amplify.
5. Analyze PCR products on agarose gels. Determine the highest dilution that still generates
a specific PCR product.
6. Set up five replicates of PCRs containing 10 µL of DNA solution from the highest dilution
level that was PCR positive, five replicates with DNA from the lowest dilution level that
was PCR negative, and five replicates with DNA from the next lowest dilution level that
was PCR negative and amplify.
7. Analyze on an agarose gel.
8. Decrease or increase the dilution level analyzed in five replicates until the pattern of
PCR results finally obtained shows a change from PCR positivity in all replicates (or the