Genotyping of Simple and Compound Short Tandem Repeat Loci Using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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• 作者：Allison P. Null ; James C. Hannis ; and David C. Muddiman
• 刊名：Analytical Chemistry
• 出版年：2001
• 出版时间：September 15, 2001
• 年：2001
• 卷：73
• 期：18
• 页码：4514 - 4521
• 全文大小：121K
• 年卷期：v.73,no.18(September 15, 2001)
• ISSN：1520-6882

文摘

The utility of electrospray ionization Fourier transform ioncyclotron resonance (ESI-FTICR) mass spectrometry asa new approach for genotyping short tandem repeats(STRs) is demonstrated. STRs are currently valued as apowerful source of genetic information with repeats thatrange in structure from simple to hypervariable. Twotetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUMTH01, a simple STR with nonconsensus alleles, and vWA,a compound STR with nonconsensus alleles. For HUMTH01, the genotype (i.e., repeat number of each allele)was determined for each of 30 individuals using massmeasurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derivedfrom heterozygous individuals were identified by mass asheteroduplexes that had formed between nonhomologousstrands. Mass measurement of the double-stranded vWAamplicon was not sufficient for determining whether theindividual was homozygous for allele subtype 18 or 18'since the amplicons differ by only 0.99 Da. Therefore,single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequentlydigesting the bottom strand using chars/lambda.gif" BORDER=0 >-exonuclease. Accuratemass measurements were obtained for the single-strandedamplicons using internal calibration and the addition ofa correction factor to adjust for the natural variation ofisotopic abundances, confirming that the individual ishomozygous for allele 18. Our results clearly demonstratethat ESI-FTICR mass spectrometry is a powerful approachto characterize both simple and compound STRs beyondthe capabilities of electrophoretic technologies.