毛干蛋白单氨基酸多态性鉴定研究

Analysis into Identification of Single Amino Acid Polymorphisms among Hair Shaft Proteins

本文建立了毛干蛋白单氨基酸多态性(single amino acid polymorphism,SAP)的质谱检测方法,以DNA测序验证方法的准确性,并设计多组样本检验SAP鉴定的重现性。对来自6个人的25份毛干样本的SAP鉴定结果进行分析,并与毛干来源人的血液外显子测序结果相比较,对于不一致位点进一步使用Sanger法测序确定SAP对应的SNP分型。共设计四组重复性实验测试,对不同直径/生长部位毛干的分型结果进行了一致性验证。通过设计的376对引物进行PCR扩增,成功检测了522个SNP分型,其中32个(6.1%)与前期外显子测序分型不一致,152个(29.1%)为外显子测序中未检测的分型,338个(64.8%)与外显子分型结果一致。四组重复性实验中,组内SAP鉴定准确率平均值为93.6%(95%CI:92.8%~94.5%),鉴定重现率平均值为96.0%(95%CI:94.3%~97.6%)。在所有25份样本中,有283个SAP位点在至少两个样本中检出,其中23个位点在全部样本中都被检出。因此,毛干蛋白SAP鉴定方法具有较高的准确性和重现性,得到的23个高检出率SAP位点可作为候选位点构建有效位点组合,呈现出潜在的法医学应用价值。

In order to confirm the mass spectrometric detection of single amino acid polymorphism(SAP)among hair shaft proteins,DNA sequencing was thereby carried out to verify the accuracy of the above SAP identification which was further validated of its reproducibility through planned multiple groups of samples.With 25 hair samples from 6 people,the related total SAPs were identified and analyzed so that their comparison was individually conducted against the sequencing of blood exons from same donor of hair shaft.Sanger sequencing was used to further test the inconsistent sites to determine the SNP typing corresponding to SAP.Four groups of repetitive experiment were contrived about hair shaft with different diameters and sprouting positions.A total of 376 pairs of primers were designed for PCR amplification of Sanger sequencing,having successfully delivered 522 SNP types,among which there were respective 32(6.1%),152(29.1%)and 338(64.8%)ones showing inconsistent,undetected and consistent to the results of the previous exon sequencing.Through comparison of the profiles of SAPs against their corresponding SNPs with the four groups of repetitive experiment,the SAP identification rendered its mean accuracy as 93.6%with the 95%CI falling into 92.8%~94.5%,and the mean reproducibility as 96.0%with the 95%CI from 94.3%~97.6%.Out of all the 25 samples,283 SAP sites were obtained and detected in no less than 2 samples,even leaving 23 ones being detected in all the tested samples.Consequently,SAP identification was verified of comparatively high accuracy and reproducibility.The 23 high reproducible SAPs might be chosen as candidate sites for a panel of effective sites to demonstrate their potential forensic value.