کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10957777 | 1099656 | 2005 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Developing the use of mismatch binding proteins for discovering rare somatic mutations
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موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیولوژی سلول
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چکیده انگلیسی
A method for detecting rare, unknown, somatic mutations could allow presymptomatic cancer screening from human fluids. Immobilized mismatch binding protein can bind DNA heteroduplexes while allowing homoduplexes to be washed away, thus enriching for rare mutations. We examined the potential use, for mutation enrichment, of a fusion protein of maltose binding protein and the mismatch binding protein TaqMutS (MBP-MutS). Unlabeled and fluorescent-labeled oligonucleotides, either perfectly complementary or with single nucleotide mismatches or deletions, were combined to form homo- or heteroduplexes that were then mixed at low ratios of hetero- to homoduplexes and enriched for heteroduplexes. Enrichment was observed using a capillary DNA sequencer. A single base deletion oligonucleotide was enriched by a factor of 29, and a mismatch oligonucleotide was enriched by a factor of 2. Nâ1 oligonucleotide synthesis fragments were enriched more than were mismatches, suggesting that these deletion fragments may compete for MutS and impede enrichment of mismatches. Purification of oligonucleotides by high pressure liquid chromatography or polyacrylamide gel electrophoresis failed to remove nâ1 fragments, thus overcoming this obstacle to enrichment of mismatch mutations may require alternative strategies, such as developing new purification methods or avoiding the use of synthetic oligonucleotides before enrichment.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Molecular and Cellular Probes - Volume 19, Issue 3, June 2005, Pages 163-168
Journal: Molecular and Cellular Probes - Volume 19, Issue 3, June 2005, Pages 163-168
نویسندگان
Larry Baum, Alex Ng, Wai Keung Leung,