Table 3

Comparison of “universal cost equivalents” of “prescreening” BRCA1 for mutations (exclusive of sequence confirmation)

PCR amplicons40 amplicons30 amplicons7 primary35 amplicons
37 secondary
PrimersStandardM13 tailedPrimary standardStandard
Secondary GC clamps
PCR reactions40, 32P labelled201 primary reaction35
4 secondary reactions
Additional pooling pre-analysis40 to 2020 to 64 to 1None
InstrumentOwl Scientific sequencing gel boxABI 377 pipetting robotCBS Scientific auto 2-D gel systemTransgenomic dHPLC system
Lanes/sample20613-150 543-151
Run time8–20 h/gel4 h/gel19 h/gel3-152 8 min/sample
% PCR reactions requiring repeat analysis5%9%13.5%3-153 <10%
  • 3-150 Products of the four multiplex groups were pooled and analysed on a single gel. Thus, the analysis requires one gel per subject.

  • 3-151 For DHPLC, 19 of the 35 amplicons are analysed at two different temperatures. Thus, analysis of 35 PCR amplicons requires 54 DHPLC runs.

  • 3-152 The gel run can now be performed in three hours (see Results).

  • 3-153 Because only individual component PCR reactions fail (rather than the whole multiplex PCR), repeats are performed with one dimensional DGGE.