MALDI-TOF based mutation detection using tagged in vitro synthesized peptides.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is a powerful method to quickly and accurately determine the masses of peptides. Most genetic analyses, however, begin with PCR amplification of a test sequence to generate DNA, which is more difficult than peptides to analyze by MALDI-TOF. We describe a method that produces a PCR product of any continuous region of coding sequence which can then be used to encode an N-terminally tagged test peptide in a coupled in vitro transcription/translation reaction. The test peptide is purified using the tag, and its mass is measured by MALDI-TOF. Truncations and amino acid substitutions in peptides coded for by the breast cancer susceptibility gene BRCA1 were readily identified using this method. The process can be multiplexed and is amenable to automation, providing an efficient, high-throughput means for mutation discovery and genetic profiling.
Garvin
A M
AM
[1] PE Biosystems, 500 Old Connecticut Path, Framingham, MA 01701. [2] Current address: Department of Biochemistry, Biocentre, University of Basel, CH-4056, Basel, Switzerlandc. alex.garvin@unibas.ch
Parker
K C
KC
Haff
L
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D019313:BRCA1 Protein / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D019398:Genes, BRCA1 / Q000235:genetics; D005820:Genetic Testing / Q000191:economics / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013557:Switzerland; D014158:Transcription, Genetic / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics
D019313:BRCA1 Protein; D010455:Peptides; D011993:Recombinant Fusion Proteins