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Clin Biochem. 2013 Oct;46(15):1572-7. doi: 10.1016/j.clinbiochem.2013.06.005. Epub 2013 Jun 20.

Rapid detection of K-, N-, H-RAS, and BRAF hotspot mutations in thyroid cancer using the multiplex primer extension.

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Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan.



The objective of this study is to develop a multiplex PCR and primer extension to detect K-, N-, H-RAS, and BRAF mutations.


DNA samples were isolated from 76 thyroid cancer patients. Multiplex amplification of exons 2 and 3 of three RAS genes and exon 15 of the BRAF gene using three pairs of primers was performed in a single tube. The products were split into three tubes. First, we used nine different-sized N-RAS and BRAF primers to detect base changes in N-RAS and BRAF. The other two tubes used seven separate different-sized K-RAS and H-RAS primers to detect base changes.


We compared these results with direct sequencing. The two methods generated identical results, but our method was superior to direct sequencing in terms of the amount of work and time involved.


We present a rapid method to detect mutations of K-, N-, H-RAS, and BRAF in human cancers.


BRAF gene; Multiplex PCR and primer extension; RAS genes; Thyroid cancer

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