A High-Throughput Mutation Detection Method Based on Heteroduplex Analysis Using Graft Copolymer Matrixes:  Application to Brca1 and Brca2 Analysis

Abstract

We present here a new approach to electrophoretic heteroduplex analysis (EHDA) based on improved matrixes. EHDA is an appealing technique for the detection of unknown point mutations because of its simplicity and high throughput. We present here a new matrix for electrophoretic heteroduplex analysis much more sensitive for insertions, deletions, and substitutions than reported for previous EHDA separations and also superior to DHPLC. This separation matrix is based on a copolymer with a comb architecture, poly(acrylamide-g-polydimethylacrylamide), made of a high molecular weight polyacrylamide backbone grafted with poly(dimethylacrylamide) side chains. The effect of operational parameters on electrophoretic resolution and sensitivity to single-nucleotide mismatches was studied using a collection of samples from patients bearing mutations in the breast cancer predisposition genes BRCA1 and BRCA2. Seventeen fragments (10 mutations), implying mostly substitutions on fragments with sizes ranging from 200 to 600 bp, were analyzed using a single set of separation conditions. A success rate of 94% was achieved with a qualitative analysis in terms of number of peaks, and 100% identification of mutations was obtained with a more quantitative test using peak width analysis. This strong improvement of performance with regard to previous HDA methods is attributed to a composite mechanism of separation, combining steric and chromatographic effects. It opens the route to a significant reduction of development time and operation cost for diagnostic and genomic applications.

EMMA, a cost- and time-effective diagnostic method for simultaneous detection of point mutations and large-scale genomic rearrangements: application to BRCA1 and BRCA2 in 1,525 patients

“…Due to the well-known limitations of capillary electrophoresis (Rozycka, et al, 2000) and topological effects (Weber, et al, 2004), false-negatives can occur in the 70bp from both extremities of the amplicon. As a result, larger and overlapping amplicons were designed so that the sequence of interest always fell outside these 70bp in at least one amplicon (Weber, et al, 2006).…”

“…Normal controls were always included in these experiments. (Weber, et al, 2004;Weber, et al, 2006) Wagner, et al, 1999) and, more recently, large-scale rearrangements using the DHPLC-derived technique called MP/LC .…”

“…Electrophoretic mobility does not only depend on the mismatch bases, but also on the local sequence environment of this mismatch, since distortions of the DNA helix at one base pair are propagated to the neighboring bases (Weber, et al, 2004). As a consequence of the above, a heteroduplex can exceptionally comigrate with the peak corresponding to the following/preceding peak in the multiplex electrophoregram.…”

“…Analysis (EMMA) is based on the use of innovative matrices increasing the electrophoretic mobility differences between homoduplex and heteroduplex DNA (Weber, et al, 2004).…”

ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy

“…Heteroduplexes and homoduplexes are separated by their different electrophoretic mobility due to their conformational differences. The resolution of EMMA is considerably better than that of standard HDA due to a combination of high resolution blockcopolymer sieving matrix, and nucleosides as additives in the electrophoretic medium [26,27]. The medium was loaded on the ABI3130XL sequencing system (Applied Biosystems 2006).…”