Hannis J C, Muddiman D C
Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA.
Rapid Commun Mass Spectrom. 2001;15(5):348-50. doi: 10.1002/rcm.234.
Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.
首次通过将流动注射分析与电喷雾电离质谱（ESI-MS）联用对聚合酶链反应（PCR）扩增子进行了表征。PCR扩增子是在人类酪氨酸羟化酶短串联重复序列位点上，从一个9.3等位基因纯合个体中扩增得到的。使用Pfu聚合酶扩增出一种产物，得到一个长度为82个碱基对（bp）的平端扩增子。第二个PCR产物是使用Taq聚合酶扩增得到的，产生了一个具有粘性末端的82 bp扩增子，末端带有单腺苷化或双腺苷化。在数据采集期间，以200 nL/min的流速，使用0.5微升定量环交替进样这两种PCR扩增子，Pfu扩增子的进样浓度为2 microM，Taq扩增子的进样浓度为1 microM。通过质量测量准确鉴定了这两种PCR扩增子，这表明ESI-MS适用于短串联重复序列的基因分型，以及对大型PCR扩增子进行高通量基因分型的潜力。
