Nucleic Acids Research, 1995, Vol. 23, No. 13 2404-2412
© 1995
METHODS |
Electrophoresis for genoptyping:temporal thermal gradient gel electrophoreisis for profiling of oiligonucleotide dissociation
1Divison of Cardiovascular Genetics, Department of Medicine, University college London Medical School, The Rayne Institute 5 UNiversity Street, London WC1E 6JJ, UK 2NHS Chemical Pathology and UNiversity Clinical Biochemistry Level D,south Laboratory Block, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, UK
*To whom corespondence should be addressed
Received April 13, 1995. Accepted May 24, 1995.
Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a singlestranded target which Is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature snapshot1 of the melting profile, allowing the distinction of perfect from imperfect base pairing. In eterozygotes, the presence of the alternative sequence must be verified wtth a second oligonucleotide complementary to the variant Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target In 20% polyacrylamlde the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the freed' rather than the ‘bound’ is displayed. The full profile of oiigonudeotide dissociation during gelelectrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks.Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate ollgcnudeotides of different melting temperatures. This provides a convenient system to examine the interaction of many different ollgonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequences) nor of oiigonudeotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene In patients with familial hypercholesterolaemia.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. J. Smith, G. Pante-de-Sousa, K. K. Alharbi, X.-h. Chen, I. N.M. Day, and K. R. Fox Combination of His-Tagged T4 Endonuclease VII with Microplate Array Diagonal Gel Electrophoresis for High-Throughput Mutation Scanning Clin. Chem., June 1, 2005; 51(6): 1043 - 1046. [Full Text] [PDF]
|
|
|
|
 |
|
 T. R. Gaunt, L. J. Hinks, H. Rassoulian, and I. N. M. Day Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping Nucleic Acids Res., May 1, 2003; 31(9): e48 - e48. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Nollau and C. Wagener Methods for detection of point mutations: performance and quality assessment Clin. Chem., July 1, 1997; 43(7): 1114 - 1128. [Abstract] [Full Text] [PDF]
|
|