No abstract

Transposing highly sensitive DNA separation methods (such as DNA sequencing with high read length or the detection of point mutations) to microchip format without loss of resolution requires fabrication of relatively long (approx. 10 cm) microchannels along with sharp injection bands. Conventional soft lithography methods, such as mold casting or hot-embossing in a press, are not convenient for fabricating long channels. We have developed a lamination-based replication technique for rapid fabrication of sealed microfluidic devices with a 10 cm long, linear separation channel. These devices are fabricated in thin cyclo-olefin copolymer (COC) plastic substrates, thus making the device flexible and capable of assuming a range of 3-D configurations. Due to the good optical properties of COC, this new family of devices combines multiple advantages of planar microfluidics and fused-silica capillaries.

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.

The potential of a series of newly synthesized poly(N,N-dimethylacrylamide) (PDMA) grafted polyacrylamide (PAM) copolymers (P(AM-PDMA)) as a replaceable separation medium for protein analysis was studied. A comparative study with and without copolymers was performed; the separation efficiency, analysis reproducibility and protein recovery proved that the P(AM-PDMA) copolymers were efficient in suppressing the adsorption of basic proteins onto the silica capillary wall. Furthermore, the size-dependent retardation of native proteins in a representative P(AM-PDMA) copolymer was demonstrated by Ferguson analysis. The results showed that the P(AM-PDMA) copolymers combine the good coating property of PDMA and the sieving property of PAM and could be applied as a sieving matrix for the analysis of native proteins.

The detection of unknown mutations is important both in population genetics research and in diagnosis. At present, two different methods must be used to detect either point mutations or large-scale genetic rearrangements, which is costly and time-consuming. We describe here a new method for the simultaneous detection of these two types of mutations. It is based on electrophoretic heteroduplex analysis (HDA) using enhanced mismatch mutation analysis (EMMA) and semiquantitative multiplexed PCR conditions. The use of such conditions allows the simultaneous search of any kind of mutation in up to five different fragments per capillary, in a single or multi-CE system. The method was validated on patient samples with mutations in the breast predisposition gene BRCA1. It leads to highly reliable and high-throughput mutation detection at low cost, as compared with classical methods.

Biallelic inactivation of the ATM gene causes ataxia-telangiectasia (A-T), a complex neurological disease associated with a high risk of leukaemias and lymphomas. Mothers of A-T children, obligate ATM heterozygote mutation carriers, have a breast cancer (BC) relative risk of about 3. The frequency of ATM carriers in BC women with a BC family history has been estimated to be 2.70%. To further our clinical understanding of familial BC and examine whether haematological malignancies are predictive of ATM germline mutation, we estimated the frequency of heterozygote mutation carriers in a series of 122 BC women with a family history of both BC and haematological malignancy and without BRCA1/2 mutation. The gene screening was performed with a new high throughput method, EMMA (enhanced mismatch mutation analysis).Amongst 28 different ATM variants, eight mutations have been identified in eight patients: two mutations leading to a putative truncated protein and six being likely deleterious mutations. One of the truncating mutations was initially interpreted as a missense mutation, p.Asp2597Tyr, but is actually a splice mutation (c.7789G[T/p.Asp2597_Lys 2643[LysfsX3). The estimated frequency of ATM heterozygote mutation carriers in our series is 6.56% (95% CI: 2.16-10.95), a significantly higher figure than that observed in the general population, estimated to be between 0.3 and 0.6%. Although a trend towards an increased frequency of ATM carriers was observed, it was not different from that observed in a population of familial BC women not selected for haematological malignancy as the frequency of ATM carriers was 2.70%, a value situated in the confidence interval of our study.Keywords Ataxia-telangiectasia Á Breast cancer Á BRCA1 Á BRCA2 Á ATM Á EMMA Electronic supplementary material The online version of this article

A new method for the detection of unknown mutations, enhanced mismatch mutation analysis (EMMA), is proposed. It is based on electrophoretic heteroduplex analysis (HDA). The resolution is considerably improved, thanks to the combination of high-resolution block-copolymer sieving matrix, and nucleosides as additives in the electrophoretic medium. The EMMA method is compared to denaturing HPLC (DHPLC) in a large-scale study of mutations in the breast cancer-associated gene BRCA2, involving 4655 DNA amplicons from 94 patients. The rate of false positives was 0.09%. The raw success rate, without optimization of the amplicons tiling, was 94%, a value much higher than that achieved earlier with HDA, and comparable with that obtained with DHPLC. An analysis of the missed mutations suggest that the success rate could be improved up to about 97%, simply by redesigning the amplicons, while retaining the speed, cost effectiveness, and simplicity of the method.

We present the routine diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis, Fluigent), a new, fast, reliable, and cost-effective method for mutation screening. This method is based on heteroduplex analysis by capillary electrophoresis and relies on the use of innovative matrices increasing the electrophoretic mobility differences between homoduplex and heteroduplex DNA, which is further enhanced by the addition of nucleosides in the separation matrix. Nucleosides interact with heteroduplex mismatched bases, hence increasing mobility difference with homoduplex. As separations are performed by multi-capillary electrophoresis, it allows for high automation, low cost, and high throughput. Moreover, EMMA, in combination with limiting PCR conditions, can be used to achieve the simultaneous detection of point mutation and large scale rearrangement in a single run.We now report on the routine diagnostic use of this method for BRCA1 and BRCA2 screening. The coding sequence and exon-intron junctions of BRCA1 and BRCA2 were amplified in 24 multiplex PCRs using a single condition. PCRs were electrophoresed with a single analytical condition on an ABI3100, and data were analyzed using dedicated software (Emmalys).The strength of this new method relies on the following assets: (1) a single condition of analysis: modeling related to melting domain is not required (2) simultaneous detection of point mutations and large scale rearrangements, (3) optimized and ready-to-use polymer that can be used on various ABI sequencers, (4) easy to use, (5) low reagent costs, and (6) throughput.