TY - JOUR
T1 - Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer
AU - Vargas, Diana Y.
AU - Kramer, Fred Russell
AU - Tyagi, Sanjay
AU - Marras, Salvatore A.E.
N1 - Publisher Copyright:
© 2016 Vargas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2016/5
Y1 - 2016/5
N2 - We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5′ anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3′ foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.
AB - We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5′ anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3′ foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.
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U2 - 10.1371/journal.pone.0156546
DO - 10.1371/journal.pone.0156546
M3 - Article
C2 - 27244445
AN - SCOPUS:84971659479
SN - 1932-6203
VL - 11
JO - PloS one
JF - PloS one
IS - 5
M1 - e0156546
ER -