Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. He...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Huidan Zhang, Jin Song, Hui Ren, Zhangrun Xu, Xiaonan Wang, Lianfeng Shan, Jin Fang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a0fa08c5e9f74e9e86b00df853f649d8
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:a0fa08c5e9f74e9e86b00df853f649d8
record_format	dspace
spelling	oai:doaj.org-article:a0fa08c5e9f74e9e86b00df853f649d82021-11-18T08:00:31ZDetection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.1932-620310.1371/journal.pone.0054510https://doaj.org/article/a0fa08c5e9f74e9e86b00df853f649d82013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23355875/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.Huidan ZhangJin SongHui RenZhangrun XuXiaonan WangLianfeng ShanJin FangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 1, p e54510 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Huidan Zhang Jin Song Hui Ren Zhangrun Xu Xiaonan Wang Lianfeng Shan Jin Fang Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
description	Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP) platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF) detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS) technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC) cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA). In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%), significantly higher than that obtained by direct sequencing (13, 13.3%). Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical samples, which will allow provision of more precise individualized cancer therapy.
format	article
author	Huidan Zhang Jin Song Hui Ren Zhangrun Xu Xiaonan Wang Lianfeng Shan Jin Fang
author_facet	Huidan Zhang Jin Song Hui Ren Zhangrun Xu Xiaonan Wang Lianfeng Shan Jin Fang
author_sort	Huidan Zhang
title	Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
title_short	Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
title_full	Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
title_fullStr	Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
title_full_unstemmed	Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
title_sort	detection of low-abundance kras mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/a0fa08c5e9f74e9e86b00df853f649d8
work_keys_str_mv	AT huidanzhang detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT jinsong detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT huiren detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT zhangrunxu detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT xiaonanwang detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT lianfengshan detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions AT jinfang detectionoflowabundancekrasmutationsincolorectalcancerusingmicrofluidiccapillaryelectrophoresisbasedrestrictionfragmentlengthpolymorphismmethodwithoptimizedassayconditions
_version_	1718422685136977920

Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

Ejemplares similares