High sensitive pool screening of point mutations in BRCA–1 using chemiluminescence detection

A nonisotopic pool-screening method of point mutations (e.g. SNP) and small insertions / deletions in genetic samples is presented. Based on a formerly described (allele-specific pool-screening, the semi nested PCR is substituted by a one-step method in combination with solid-phase technique and chemiluminometric detection [3].

The allele-specific PCR was designed to discriminate between T and G changing the amino acid 300 from Cysteine to Glycine in the breast cancer gene 1 (BRCA–1).

The PCR products were coupled via their Biotin label to Streptavidin-coated microtiter plates. For chemiluminometric measurement an A(lcaline)-P(hosphatase)-labeled monoclonal anti-Digoxigenin-antibody was added. The antibody detects the PCR products resulting out of the amplification of the mutant allele. AMPPD with amplifier was used as the chemiluminescent substrate for AP [1–2].

The method is able to detect one mutant allele in a total of 2048 alleles. Pool sizes from 32 up to 128 samples are practicable (=64 to 256 alleles). The signal-to-noise-ratio is better than 3: 1 vs. cut-off for pools with 128 specimens.

The method is very useful in following applications:

• for population studies, especially with rare mutations

• for determination of allele frequencies in a population

Furthermore the use of microtiter plates allows an automated execution of the post PCR steps i) PCR product extraction, and ii) detection & measurement of the mutant allele. The method is able to reduce the costs about 60 to 90% for PCR per sample (depending on pool size).

BRCA-1 - Breast Cancer - Chemiluminescence - Dioxetane - Light Reaction - PCR - Poolscreening