PCR-Based Methods for the Enrichment of Minority Alleles and Mutations

Abstract

BACKGROUND:The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze.CONTENT: Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change.

Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the Adenomatous Polyposis Coli Gene

“…The aforementioned methods usually require nanogram amounts of mutated DNA. Altogether, these data support the need of an additional enrichment step to reach high sensitivity as was already postulated (32).…”

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

“…Furthermore, highly sensitive methods are required for the detection and identification of somatic mutations in early tumorigenesis and the emergence of resistance mutations (Milbury, et al, 2009b). Traditional Sanger sequencing has been considered the gold standard for detecting and identifying genetic alterations.…”

Mutant Enrichment with 3′-Modified Oligonucleotides

“…Such subtle differences, however, may not provide clear discrimination between wild-type and mutant DNA, which is responsible for the low enrichment rate and difficulty in adequate thermal control. 3 The PNA/LNA-mediated PCR clamping technique is, in a sense, a method based on thermal differentiation. PNA is a DNA mimic in which the phosphoribose backbone is replaced by a peptide-like repeat of 2-aminoethyl-glycine units.…”

“…In principle, this does not aberrantly alter the wild-type sequences and leads to much lower false-positive rates. 3 The PNA/LNA-mediated clamping technique has been used for over a decade and is an attractive method that has proven to be potentially applicable to many situations, including the examination of various cancer mutations, 8,19 -23 identification of viral or bacterial variants, 24,25 genotyping for single-base substitutions, 26,27 detection of low-level somatic mosaicism 28 or mitochondrial heteroplasmy, 29,30 and prenatal diagnosis via maternal plasma, 31 among many others. However, the high cost and limited availability have restricted its usage.…”

“…Sanger sequencing, the robust standard method, can only detect approximately 20% of mutant alleles in a background of normal alleles, whereas other, more sensitive, PCR-based assays such as restriction fragment length polymorphism, amplification refractory mutation system PCR, denaturing high-performance liquid chromatography, and real-time PCR are more limited, with accuracies below a certain level of allele minority. 3 Amplification refractory mutation system PCR, the most widely used method, is an amplification strategy in which a PCR primer is designed to discriminate among templates that differ by a single nucleotide residue. This method is simple and time efficient but sometimes produces serious false-positive results because the mutantspecific primer may induce false sequences.…”