Laser desorption mass spectrometry for point mutation detection
- Oak Ridge National Lab., TN (United States); and others
A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.
- OSTI ID:
- 598193
- Journal Information:
- Genetic Analysis: Biomolecular Engineering, Vol. 13, Issue 4; Other Information: PBD: Oct 1996
- Country of Publication:
- United States
- Language:
- English
Similar Records
Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene
GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations