Abstract
SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.
Highlights
Ripped apart than the perfectly complementary mutant foot hybrids
We have found that the inclusion of tetramethylammonium chloride (TMAC) in the PCR assay buffer tends to stabilize the perfectly complementary mutant foot hybrids, and tends to destabilize the mismatched wild-type foot hybrids, especially if the mismatch occurs at the 3′ nucleotide of the foot of the SuperSelective primer[4]
When ten or fewer target molecules are present in a sample, it is not clear whether the resultant late arising signal is due to the presence of those rare mutant target molecules, or whether the resultant late arising signal is due to the undesired amplification of the abundant closely related wild-type molecules
Summary
Ripped apart than the perfectly complementary mutant foot hybrids. there is a significantly greater probability that a DNA polymerase will find and bind to a mutant foot hybrid and initiate exponential synthesis before that foot hybrid dissociates, than the considerably lower probability that a DNA polymerase will initiate synthesis on a very short-lived mismatched wild-type foot hybrid. We have found that the inclusion of tetramethylammonium chloride (TMAC) in the PCR assay buffer tends to stabilize the perfectly complementary mutant foot hybrids, and tends to destabilize the mismatched wild-type foot hybrids, especially if the mismatch occurs at the 3′ nucleotide of the foot of the SuperSelective primer[4] These PCR assays are carried out with one SuperSelective primer and a conventional reverse primer for the amplification of rare DNA fragments possessing the target mutation, and the results enable those rare target molecules to be detected in the presence of abundant closely related wild-type molecules[3]. Utilizing samples containing DNA fragments from 10,000 human genomes, we showed that assays containing SuperSelective primer pairs reliably provide truepositive signals in samples containing as little as five mutant DNA target fragments, yet do not produce falsepositive signals when mutant DNA target fragments are not present in the sample
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