Abstract A23: Rare allele enrichment by DNA melting analysis
Authors: Zhou Luming , Wittwer Carl 
Published: June 2012

Since the sequencing of the human genome and the development of high throughput genotyping techniques, many genes have been identified that are associated with a variety of diseases through their SNPs and mutations. For example, in lung cancer, mutations in the gene encoding epidermal growth factor reporter (EGFR) can confer either positive therapeutic response or drug resistance to tyrosine kinase inhibitors. Additionally, mutations in the BRAF gene are strongly associated with cancerous thyroid nodules. In the early and post-treatment stages of cancer or in prenatal diagnostics, the proportion of the mutant allele is typically low within a background of wild-type. Even after amplification by standard PCR, the low levels of the minority allele that are present in tumor tissue or in plasma circulating DNA are not sufficient to permit genotyping or sequencing. Mutation detection plays a key role in several areas of medicine including diagnosis, treatment and prognosis. In cancer-related gene mutation diagnostics, low-level (&lt;10%) mutations cannot be sequenced by standard PCR or genotyping techniques. We have developed methods of rare allele enrichment and melting curve detection using Snapback primers and unlabeled probes.

A snapback primer is a tail at the 5′ end of a primer that is complementary to its own extension product. When intramolecular hybridization is favored over intermolecular pairing, a hairpin is formed whose stem melting behavior depends on sequence. Rapid cycle PCR with a short extension time (0 sec) and an extension temperature lower than the Tm of the hairpin of the wild-type, perfectly matched allele obstructs primer extension while mismatched alleles denature, thus providing the competitive advantage and preferential amplification of minority alleles. Minority alleles down to 0.1% (1:1000) can be detected.

Even greater detection sensitivity can be achieved using allele specific PCR, probe blocking and melting analysis. Unlabeled probes, dual hybridization probes, or molecular beacons can be used. Allele-specific competitive blocker PCR (ACB-PCR) was modified by using asymmetric PCR and melting analysis to increase the sensitivity. The probe serves as both a blocker and produces the probe melting curve to implement detection. Mutation detection by probe melting curve analysis is more reliable than PCR quantification because only the specifically amplified allele produces the melting curve associated with the target. Quantitative PCR with dsDNA dyes cannot distinguish the intended PCR product from primer dimers and other non-specific PCR products. In contrast, when using probe melting curves to detect mutations, the melting temperature and overall melting profile of probe melting curves specifically identify the known mutant target. Additionally, based upon probe melting temperatures of wild-type and mutant hybrids, a precise annealing temperature between these Tms guarantees that the PCR selectively amplifies the minority allele. Low allele specific primer concentrations (asymmetric PCR) further increase the sensitivity and increase the probe signal allowing a sensitivity of 0.001% (1:105) mutant to wild-type genomic DNA, or just a single copy in a 500 ng genomic DNA.

Methods that do not require labeled probes (snapback primer and unlabeled probes) can be used to selectively amplify minority alleles by methods that are rapid (less than 20 minutes), easy to design, inexpensive and reliable.

Citation Information: Cancer Prev Res 2011;4(10 Suppl):A23.