Heteroduplex Analysis Matrices

At its earliest inception, the sensitivity of HA was limited by the availability of suitable gel matrices. Slab-gel systems are limited to agarose and polyacrylamide. The improved MDE gel solution (Cambrex) is a polyacryl-amide-like matrix with a high sensitivity for DNA conformational changes. The gel's unique structure allows DNA separation on the basis of both size and conformation, thus increasing the probability of detecting sequence differences from as low as 15% achieved in standard polyacrylamide gels to approximately 80%.

A number of modifications to the matrix have been reported to improve the gel's ability to enhance fragment retardation. For example, the use of mild denaturants which exaggerate the effects of the mismatch forms the basis of conformation-sensitive gel electrophoresis (CSGE).[6-8] Ganguly et al.[9] have adapted the technique for use with fluorescently labeled fragments which can then be resolved using the ABI 377 (Applied Biosystems) DNA sequencer platform. They labeled PCR products with either 6-FAM, HEX, or NED prior to heteroduplex formation.

An array of matrices is now available for use with capillary electrophoresis such as cellulose derivatives and hydrophilic polymers substantially improving the resolution of HA.[10-12] One of the earliest reports to describe

A. Bubble heteroduplex

B. Bulge heteroduplex

Fig. 1 Types of heteroduplex. (A) ''Bubble-type'' heteroduplex forms in the presence of one or more base substitutions. (B) ''Bulge-type'' heteroduplex forms with larger lesion such as insertion or deletion. (View this art in color at www.dekker.com.)

Fig. 1 Types of heteroduplex. (A) ''Bubble-type'' heteroduplex forms in the presence of one or more base substitutions. (B) ''Bulge-type'' heteroduplex forms with larger lesion such as insertion or deletion. (View this art in color at www.dekker.com.)

the adaptation of HA to capillary electrophoresis employed an entangled polymer matrix under nondenatur-ing conditions.1-13-1 Rozycka et al.[14] adapted the technique for use with a single capillary fragment analyzer (ABI 310, Applied Biosystems) and were able to achieve high sensitivity in products of <350 bp. Such systems utilize replaceable linear polyacrylamides (LPAs) offering lower viscosity, high resolution, and rapid replacement of matrix thus permitting multiple reuse of the capillary. To attain high-throughput coupled with a greater resolution, Gao and Yeung[15] described temperature-gradient capillary electrophoresis. They exploited the lower melting temperature of heteroduplexes relative to homoduplexes and applied a temperature gradient to partially denature the DNA fragments during electrophoresis. By covering the entire range of melting temperatures of duplexes, they were able to achieve a high sensitivity for single-base substitutions set in a 96-well format. Our group, in an effort to raise the level of throughput without compromising sensitivity, designed a modification called multiplex capillary heteroduplex analysis (MCHA) using the 96-capillary MegaBACE1000 DNA Analyzer platform (Amersham Biosciences).1-16-1 We described an additional level of throughput achieved by multiplexing up to six differently labeled (6-FAM, HEX, or TET) or sized products (up to 494 bp) in a single well. This coupled with 96-sample capability per run allows up to 576 samples in 1 hr or as many as 3000 samples in a day. Multiplex capillary heteroduplex analysis is extremely effective at detecting known alterations (specific) but has proven to be a useful scanning tool for the detection of novel mutations (Fig. 2).

et al.[17] compared conventional horizontal gel HA with SSCP using known mutations from four different genes. Heteroduplex analysis detected 93% of mutations compared with 67% using SSCP; however, when combined, all mutations were revealed.

A comparison of HA, SSCP, and DGGE used for detecting apolipoprotein mutations in a large cohort of patients with hypercholesterolaemia revealed HA and DGGE to be equally as effective but superior to SSCP.[18]

Recently, Crepin et al.[19] designed an assay combining HA and an SSCP variation they called mutation detection gel analysis (MDGA) for the detection of multiple mutations in MEN1 using fluorescently labeled PCR fragments. Heteroduplexes were resolved on a 0.5 x MDE gel and for MDGA, single-stranded products on an Elliomut gel (Elliotek, Paris), both electrophoresed as a slab using an automated sequencer (ABI 377). Peaks were compared with controls and abnormal profiles subsequently sequenced to reveal the true nature of any underlying mutation. They demonstrated that each mutation cast a specific and reproducible pattern dependent on the base change. Heteroduplex analysis detected 88.5% of mutations and 100% when combined with MDGA. The authors questioned the specificity of the tests and suggested this be remedied by reducing the fragment length to less than 400 bp. They concluded that even if the positive predicted value of the combined test does not reach 100%, the negative predictive value does, a point of critical importance in genetic testing.

The study of Hoskins et al.[16] which tested 12 sequence changes (2 deletions and 10 substitutions) in four Bardet-Biedl syndrome genes concluded that HA when adapted for use on a 3% nondenaturing capillary injected matrix (LPA) gave outstanding sensitivity (100%) alone. Furthermore, they calculated the sample cost to be at least one-tenth that for direct sequencing.

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