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An automated fluorescent single strand conformation polymorphism technique for high throughput mutation screening

Weng, Jianping; Lehto, Markku; Berglund, Anna and Groop, Leif LU (2001) In Chinese Medical Journal 114(11). p.1147-1150
Abstract
OBJECTIVE: To develop a high throughput mutational detection method by multiple fluorescence-labeled polymerase chain reaction (PCR) products. METHODS: A total of 27 known mutations including 22 substitutions, 3 insertions (1, 2 and 7 bp) and 2 deletions (1 and 2 bp) in the hepatocyte nuclear factor (HNF)-4 alpha, glucokinase and HNF-1 alpha genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in non-denaturing gel conditions. RESULTS: Twenty-five of 27 variants (93%) could be detected by combining 5% glycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%)... (More)
OBJECTIVE: To develop a high throughput mutational detection method by multiple fluorescence-labeled polymerase chain reaction (PCR) products. METHODS: A total of 27 known mutations including 22 substitutions, 3 insertions (1, 2 and 7 bp) and 2 deletions (1 and 2 bp) in the hepatocyte nuclear factor (HNF)-4 alpha, glucokinase and HNF-1 alpha genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in non-denaturing gel conditions. RESULTS: Twenty-five of 27 variants (93%) could be detected by combining 5% glycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%) variants were identified using 5% glycerol and 10% sucrose conditions, respectively. CONCLUSION: This fluorescence-based PCR single strand conformation polymorphism technique represents a simple, non-hazardous, time-saving and sensitive method for high throughput mutation detection. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Chinese Medical Journal
volume
114
issue
11
pages
1147 - 1150
publisher
Chinese Medical Association
external identifiers
  • pmid:11729508
  • scopus:0035177561
ISSN
0366-6999
language
English
LU publication?
yes
id
b2418db6-e348-4ce5-b2ea-c109c6e24226 (old id 1122517)
alternative location
http://www.cmj.org/Periodical/PDF/2001/2001111147.pdf
date added to LUP
2008-07-17 11:20:43
date last changed
2018-01-07 09:02:29
@article{b2418db6-e348-4ce5-b2ea-c109c6e24226,
  abstract     = {OBJECTIVE: To develop a high throughput mutational detection method by multiple fluorescence-labeled polymerase chain reaction (PCR) products. METHODS: A total of 27 known mutations including 22 substitutions, 3 insertions (1, 2 and 7 bp) and 2 deletions (1 and 2 bp) in the hepatocyte nuclear factor (HNF)-4 alpha, glucokinase and HNF-1 alpha genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in non-denaturing gel conditions. RESULTS: Twenty-five of 27 variants (93%) could be detected by combining 5% glycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%) variants were identified using 5% glycerol and 10% sucrose conditions, respectively. CONCLUSION: This fluorescence-based PCR single strand conformation polymorphism technique represents a simple, non-hazardous, time-saving and sensitive method for high throughput mutation detection.},
  author       = {Weng, Jianping and Lehto, Markku and Berglund, Anna and Groop, Leif},
  issn         = {0366-6999},
  language     = {eng},
  number       = {11},
  pages        = {1147--1150},
  publisher    = {Chinese Medical Association},
  series       = {Chinese Medical Journal},
  title        = {An automated fluorescent single strand conformation polymorphism technique for high throughput mutation screening},
  volume       = {114},
  year         = {2001},
}