Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene.

Abstract

We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.

The detection of K-ras mutations in colorectal cancer using the amplification-refractory mutation system

“…Detection levels of mutant ras sequences present at as low as 1 in 105 wild-type sequences have been reported (Ehlen and Dubeau, 1989;Cha et al, 1992) and 1 in 103 (this study and Carpenter et al, 1996). This ability of the technique to detect rare mutations in a background of normal DNA demonstrates its potential role in screening or in the monitoring of residual disease.…”

“…In fact, control reactions for tumour DNA analysis had routinely lower ratios of mutant to wild-type input DNA. Other groups (Ehlen and Dubeau, 1989;Stork et al, 1991;Cha et al, 1992;Hasegawa et al, 1995;Hayashi et al, 1995;Carpenter et al, 1996) have reported tests based on the same principles as the ARMS tests described here.…”

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

“…Probes speci®c for each point mutation can interfere with one another during hybridization or polymerase extension. Thus, while allele-speci®c PCR can detect individual point mutations, it is not e ective for multiplex detection of all possible K-ras mutations in a homogeneous assay (Cha et al, 1992;Hasegawa et al, 1995;Hayashi et al, 1994Hayashi et al, , 1995Losi et al, 1992;Tada et al, 1993;Wu et al, 1989).…”

“…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.…”

“…K-ras mutations may be detected in the primary tumor by direct DNA sequencing, allele-speci®c oligonucleotide hybridization, or restriction digest techniques (Chen and Viola, 1991;Di Giuseppe et al, 1994;Kahn et al, 1991;Levi et al, 1991;Mitsudomi et al, 1991). In addition, several research groups have used high sensitivity techniques such as phage cloning, allelespeci®c PCR, or repetitive restriction digests to detect K-ras mutations in stool or lymph nodes of cancer patients as indicators of early disease or micrometastases (Caldas et al, 1994;Cha et al, 1992;Haliassos et al, 1989;Hasegawa et al, 1995;Hayashi et al, 1994Hayashi et al, , 1995Jacobson and Moskovits, 1992;Mansfeld and Bos, 1992;Nollau et al, 1996;Orita et al, 1989;Ravin-Smith, 1995;Rust et al, 1993;Sidransky et al, 1992;Tada et al, 1993;Westra et al, 1993). However, the techniques utilized for K-ras detection have limitations.…”