Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational anaylsis

Abstract

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

“…Several PCR based techniques can detect single-base mutations present in a minority population of human tumor cells: allele-speci®c PCR (AS-PCR), which includes MASA, PASA, MS-PCR, MAMA or ARMS ampli®cation (Cha et al, 1992;Chehab and Kan, 1989;Hasegawa et al, 1995;Hayashi et al, 1994Hayashi et al, , 1995Kwok et al, 1990;Li et al, 1990;Lo et al, 1991;Mansfeld and Bos, 1992;Orita et al, 1989;Ruano and Kidd, 1989;Rust et al, 1993;Seyama et al, 1992;Tada et al, 1993;Wu et al, 1989) as well as by primer mediated RFLP (Chen and Viola, 1991;Di Giuseppe et al, 1994;Kahn et al, 1991;Levi et al, 1991;Mitsudomi et al, 1991) or electrophoretic mobilitybased methods (Chen and Thilly, 1994;Fodde and Losekoot, 1994;Khrapko et al, 1994). However, these ampli®cation methods are susceptible to false-positive signals due to mis-extension of the mutant-speci®c primer on wild-type target.…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…al in 1994 15 . The same method of allele separation was first demonstrated on a commercial capillary DNA sequencing in 2001 73,108 .…”

“…Mitochondrial mutations-In Khrapko et al 15,38,[55][56][57] , Coller et al 39 and Marcelino et al 58 the combination of high fidelity DNA amplification and constant denaturing capillary electrophoresis was used to discover the point mutations distributed in a 100 bp mitochondrial DNA sequence in human DNA replication by human DNA polymerase gamma, cells in culture, various tissues and derived tumors. Their work demonstrated for the first time that treatment of human cells with a chemical mutagen induced a reproducible set of mutations in mitochondrial DNA, that the spectrum of the "spontaneous" point mutations found in human cell cultures were not significantly different from those found in multiple human tissues and their derived tumors and that this in vivo mutational spectrum in the lung was not affected by cigarette smoking.…”

“…The improved methods have included modes of sequencing DNA [5][6][7] , allele-specific recognition of mutant sequences [8][9][10] and methods that separate point mutant from wild type sequences on the basis of lower thermal stability in wild type/mutant heteroduplexes as opposed to wild type homoduplexes [11][12][13][14][15][16][17][18][19] . A review of all of these contributions is beyond the scope of this effort; here we focus on the technical history and applications of methods that depend on the separation of wild type and mutant sequences on the basis of kinetics of DNA melting and reannealing while DNA is moved by electromotive force through a molecular sieve such as a polyacrylamide gel.…”

“…This observation led us to combine the method of capillary gel electrophoresis that had been developed in Barry Karger's laboratory at Northeastern University because the replaceable capillary matrices did not use a crosslinking agent 31,32 . This development combined with efforts led by Konstantin Khrapko at MIT increased peak detection sensitivity to < 1% by decreasing background noise from nonspecific gel binding and increasing peak resolution via the intrinsic qualities of capillary electrophoresis 15 . Laser induced fluorescence, also adopted from Karger's laboratory 31,33 , was used to detect migrating peaks replacing the cumbersome methods of detection of 32 Plabelled DNA.…”