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

PCR Detection of Tumor Cells 189
Fig. 2. Consensus PCR using primers FR3A and LJH. In 5 of 16 cases, the consensus PCR
product of the malignant clone can be distinguished from the surrounding polyclonal smear.
In all other cases, only a smear, like the one seen in buffy coat DNA, is visible. This figure
also illustrates the low rate of distinct consensus PCR products obtained if using DNA instead
of RNA. Detection rates can further be increased by using two different consensus strategies
in parallel.
5. Scrape layer of bacteria from the plate and suspend in 100 µL of lysis buffer. Vortex
vigorously.
6. Incubate the suspension at 65°C for 15 min.
7. Inactivate proteinase K at 95°C for 15 min.
8. Pellet debris by centrifugation at maximum speed and 4°C. Transfer the supernatant
containing the plasmids to a fresh tube. Store at 4°C.
9. Analyze plasmids for inserts by PCR using primers complementary to the M13 sites
of the vector. Set up a PCR containing 5 µL of 10× PCR buffer, 2 mM MgCl 2 , each
deoxynucleotide at 0.1 mM, primers M13 forward plus M13 reverse at 0.4 µM each, 2.5 U
Taq-DNA-polymerase, and 1 µL of plasmid solution. Amplify for 40 cycles of 1-min
denaturation at 94°C, 1 minute annealing at 65°C, and extension at 72°C, followed by a
final extension of 5 min at 72°C. Analyze on a 2% agarose gel.
3.3.2. Sequencing and Identification of the CDR Regions of the Myeloma Clone
1. Prepare sequencing reactions using commercially available kits according to the manufacturer’s
instructions with 4 to 8 µL of plasmid solution, and perform cycle sequencing.
2. Analyze on a sequencer, for example Alf-express (Pharmacia, Freiburg, Germany).
3. Analyze obtained sequences by aligning them to known CDR regions (examples for CDR3
regions are given in Fig. 3; see Note 2).
4. Identify the consensus PCR product of the malignant clone by its predominant occurrence
among the polyclonal CDR-regions. B-cells of normal clones are not encountered more
frequently than once in 20000 B-cells (12). At least five identical clones should be typed
to identify the myeloma clone.