[Application of denaturing high performance liquid chromatography to mutation detection of the c-kit gene in mastocytosis]
[Application of denaturing high performance liquid chromatography to mutation detection of the c-kit gene in mastocytosis]
The aim of study was to detect mutation of tyrosine domain of c-kit gene in mastocytosis using denaturing high performance liquid chromatography technique, and to investigate the significance of this gene mutation in diagnosis and therapy of mastocytosis. Genomic DNA was obtained from bone marrow or peripheral blood leukocytes using the phenol/chloroform method from 7 mastocytosis patients. PCR was performed with AmpliTaq Gold DNA polymerase and 100 ng genomic DNA to amplify the entire coding sequence and exon-intron boundaries of c-kit exon 17 approximately exon 19. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE DNA Fragment Analysis System. Each PCR product was mixed with an equal quantity of amplified human placental DNA (served as normal control) and was denatured at 95 degrees C for 5 minutes, followed by slowly cooling down to room temperature by 1.5 degrees C per minute to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator. Samples with extra peaks or with different peak form on DHPLC were directly sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and ABI 3100 Genetics Analyser. The results showed that DHPLC revealed an aberrant peak in one patient in exon 17 and the D816V mutation was identified by direct sequencing. The other two patients had an extra peak for exon 18/19 and direct sequencing revealed a conservative sequence change (L862L) within exon 18. It is concluded that denaturing high performance liquid chromatography is a high efficiency and reliable technique for mutation detection of c-kit gene and the detection results would be helpful for the selection of therapy in mastocytosis.
analysis, proto-oncogene proteins c-kit, proto-oncogene proteins, chromatography, base sequence, diagnosis, humans, bone marrow, dna, protein, aged, therapy, china, mutation, adult, bone, proteins
981-984
Zhang, Ling-Yan
f7a0ee18-f420-46a2-88fa-cff6aa7adbe7
Xu, Gong-Li
e0bf7fa9-485c-4045-968b-8a8c689184ab
Cross, Nicholas C.P.
f87650da-b908-4a34-b31b-d62c5f186fe4
October 2006
Zhang, Ling-Yan
f7a0ee18-f420-46a2-88fa-cff6aa7adbe7
Xu, Gong-Li
e0bf7fa9-485c-4045-968b-8a8c689184ab
Cross, Nicholas C.P.
f87650da-b908-4a34-b31b-d62c5f186fe4
Zhang, Ling-Yan, Xu, Gong-Li and Cross, Nicholas C.P.
(2006)
[Application of denaturing high performance liquid chromatography to mutation detection of the c-kit gene in mastocytosis].
Zhongguo shi yan xue ye xue za zhi [Journal of Experimental Hematology], 14 (5), .
Abstract
The aim of study was to detect mutation of tyrosine domain of c-kit gene in mastocytosis using denaturing high performance liquid chromatography technique, and to investigate the significance of this gene mutation in diagnosis and therapy of mastocytosis. Genomic DNA was obtained from bone marrow or peripheral blood leukocytes using the phenol/chloroform method from 7 mastocytosis patients. PCR was performed with AmpliTaq Gold DNA polymerase and 100 ng genomic DNA to amplify the entire coding sequence and exon-intron boundaries of c-kit exon 17 approximately exon 19. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE DNA Fragment Analysis System. Each PCR product was mixed with an equal quantity of amplified human placental DNA (served as normal control) and was denatured at 95 degrees C for 5 minutes, followed by slowly cooling down to room temperature by 1.5 degrees C per minute to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator. Samples with extra peaks or with different peak form on DHPLC were directly sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and ABI 3100 Genetics Analyser. The results showed that DHPLC revealed an aberrant peak in one patient in exon 17 and the D816V mutation was identified by direct sequencing. The other two patients had an extra peak for exon 18/19 and direct sequencing revealed a conservative sequence change (L862L) within exon 18. It is concluded that denaturing high performance liquid chromatography is a high efficiency and reliable technique for mutation detection of c-kit gene and the detection results would be helpful for the selection of therapy in mastocytosis.
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Published date: October 2006
Keywords:
analysis, proto-oncogene proteins c-kit, proto-oncogene proteins, chromatography, base sequence, diagnosis, humans, bone marrow, dna, protein, aged, therapy, china, mutation, adult, bone, proteins
Identifiers
Local EPrints ID: 60463
URI: http://eprints.soton.ac.uk/id/eprint/60463
ISSN: 1009-2137
PURE UUID: 394b6d6b-6e7d-45e7-a25f-fdcccfa881b2
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Date deposited: 09 Sep 2008
Last modified: 09 Jan 2022 03:08
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Author:
Ling-Yan Zhang
Author:
Gong-Li Xu
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