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Vol 46(2012) N 3 p. 414-420;
I.V. Botezatu1, K.I. Zhordania1, A.I. Karseladze1, A.M. Stroganova1, V.N. Kondratova1, V.P. Shelepov1, M.V. Telkov2, A.V. Lichtenstein1*

Scanning of Mutations in Short Amplicons: Optimization of DNA Melting Method

1Blokhin Russian Cancer Research Center of the Russian Academy of Medical Sciences, Moscow 115478 Russia
2BIO-RAD Laboratory, Moscow 117105 Russia


*alicht@mail.ru
Received - 2011-10-23; Accepted - 2011-11-21

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (post-amplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the opentube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to 'Sanger' sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.

DNA melting, mutations, K-RAS, PCR, cancer, HRMA



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