Hall Thomas A, Sannes-Lowery Kristin A, McCurdy Leslie D, Fisher Constance, Anderson Theodore, Henthorne Almira, Gioeni Lora, Budowle Bruce, Hofstadler Steven A
Ibis Biosciences, Abbott Molecular, Inc., Carlsbad, California 92008, USA.
Anal Chem. 2009 Sep 15;81(18):7515-26. doi: 10.1021/ac901222y.
We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.
我们描述了一种基于多重聚合酶链反应（PCR），随后进行去溶剂化并使用电喷雾电离质谱（PCR/ESI-MS）进行直接分析的用于人类线粒体DNA（mtDNA）高分辨率分析的自动化系统。该检测方法使用24对引物扩增mtDNA控制区域中的靶点，包括法医分析中通常测序的高变区域。由产物碱基组成构成的图谱可以存储在数据库中，相互比较，并与测序结果进行比较。通过该技术保留了约94%通过测序获得的鉴别信息。该检测方法比测序最小的HV1和HV2区域更具鉴别力，因为它检测了更多的线粒体基因组。与群体数据库相比的图谱可以采用用于评估一致的mtDNA序列显著性的相同统计方法。该检测方法不受长度异质性的阻碍，可以直接分析模板混合物，并且每个反应对总DNA的灵敏度<25 pg。在28个月内对同一样本进行的3331次独立试验分析产生的平均质量测量不确定度为10.1±8.0 ppm，超过99%的试验通过自动化分析产生完整图谱。该技术可直接应用于法医生物学证据的分析。
