Improved detection of BRAF V600E using allele-specific PCR coupled with external and internal controllers.

Sci Rep 2017 10 23;7(1):13817. Epub 2017 Oct 23.

Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.

Although traditional allele-specific PCR (tAS-PCR) is a common screening method for BRAF V600E mutations, its lower amplification specificity and mutation selectivity have limited its clinical applications. We hypothesize that these limitations are associated with the weaker specificities of allele-specific primers and the thermodynamic driving forces of DNA polymerase. We used three strategies to circumvent these limitations, namely, modifying allele-specific primers, introducing a competitive external allele-specific controller (i.e., cAS-PCR), and introducing a referenced internal positive controller in the cAS-PCR (i.e., rcAS-PCR). The amplification sensitivities and specificities were influenced by the position of the artificially introduced mismatched nucleotide in the allele-specific primers. Moreover, both cAS-PCR and rcAS-PCR could detect single-copy BRAF V600E alleles with higher mutation selectivity (0.1%) than tAS-PCR. In addition, cAS-PCR eliminated false-negative results caused by various PCR inhibitors that might be present in the DNA solutions. The rcAS-PCR could also be employed to avoid the false-negative results caused by low-abundance input templates in cAS-PCR. In conclusion, rcAS-PCR provides a rapid, simple, and low-cost method for detecting low levels of the mutated BRAF V600E gene.

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Source
http://www.nature.com/articles/s41598-017-14140-2
Publisher Site
http://dx.doi.org/10.1038/s41598-017-14140-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653796PMC
October 2017
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References

(Supplied by CrossRef)

W Chen et al.
CA Cancer J Clin 2016

E Cutsem Van et al.
J Clin Oncol 2007

D Cunningham et al.
N Engl J Med 2004

F Nicolantonio Di et al.
J Clin Oncol 2008

S Benvenuti et al.
Cancer Res 2007

S Siena et al.
J Natl Cancer Inst 2009

R Wong et al.
J Clin Oncol 2008

CA Milbury et al.
Clin Chem 2009

CA Milbury et al.
Clin Chem 2009

F Xie et al.
Clin Biochem 2014

MM Machnicki et al.
Acta Biochim Pol 2013

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