LASER-DESORPTION MASS-SPECTROMETRY FOR POINT MUTATION DETECTION

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 poi nt 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 w ork, two different approaches were pursued to detect G551D point mutat ion in the cystic fibrosis gene. The strategy is to amplify the desire d region of DNA template by PCR using two primers that overlap one bas e 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 size d primers that match the mutant sequence et DNA, an abnormal PCR produ ct will be produced. Thus, the mass spectrometer can be used to identi fy 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 use d to detect the length of the restriction fragments generated by diges tion of a PCR generated target fragment.