Determination of Hereditary Mutations in the BRCA1 Gene Using Archived Serum Samples and Capillary Electrophoresis

Abstract

Analysis of DNA variation in biological samples most frequently utilizes the polymerase chain reaction (PCR) performed on extracted genomic DNA, followed by visualization of alleles using various methodologies. Few reports have demonstrated that amplification of DNA from plasma and serum samples is possible. We have performed DNA amplification on a large set of serum samples (n = 2955). Here, we report that known hereditary mutations in the BRCA gene can efficiently be analyzed in serum samples collected and stored over several decades. Fragments were PCR-amplified following a short initial denaturation of the serum sample in a standard microwave oven. Fragment analysis was subsequently performed using a DNA capillary-sequencing instrument. The PCR success rates were fragment- and size-dependent ranging from 83.2% to 97.9%. Of the 11,820 polymerase chain reactions performed, the overall PCR success rate was 91.3% (10,796/11,820), which is comparable to PCR performed on genomic DNA. The advantage of the method described herein is its ability to utilize archival material stored in serum biobanks for long periods of time.

Investigation of Established Genetic Risk Variants for Glioma in Prediagnostic Samples from a Population-Based Nested Case–Control Study

“…The samples were stored at À25 C for up to 40 years. The samples were genotyped by means of cycling temperature capillary electrophoresis (23), a PCR-based method that has successfully been applied in previous projects investigating Janus serum samples (24,32). Because the DNA is expected to be degraded, we applied a primer design that yielded short amplicons (<100 bases).…”

“…Because the DNA is expected to be degraded, we applied a primer design that yielded short amplicons (<100 bases). This approach has proven to achieve a PCR success rate similar to that seen when using full-length DNA (24). In addition to using a method well suited for the samples at hand, cases and controls were matched for date of sample collection (i.e., storage time).…”

“…Five microliter aliquots of serum was transferred into 96-well microplates (Axygen; VWR) and subjected to enzymatic amplification, as described by Ekstrøm and colleagues (24). Serum aliquots were denatured at 95 C for 8 minutes.…”

BRCA1 mutations in ovarian cancer and borderline tumours in Norway: a nested case–control study

“…We have previously reported that known hereditary mutations in the BRCA gene can efficiently be analysed in serum samples (Ekstrm et al, 2004). With a primer design producing less than 100 bp products, we were able to achieve amplification success rates ranging from 83 to 98%, depending on primer pair.…”

“…A detailed description of all methodological aspects is published elsewhere (Ekstrm et al, 2004). Briefly, 5 ml of serum were aliquoted to 96-well plates (Axygen, Tamro Medlab AS, Oslo, Norway), followed by microwave boiling at 1200 W for 4 min.…”