用于复杂亲缘关系分析的分子标记组合、引物组、试剂盒及分析方法

本发明涉及个体身份识别，尤其是涉及一种用于复杂亲缘关系分析的分子标记组合、引物组、试剂盒及分析方法。

背景技术：

1、复杂亲缘关系分析是法医物证学学科发展的重要研究方向，在司法实践、刑事侦查、灾害遇难者身份识别等领域具有重大社会需求。目前，亲子鉴定和全同胞鉴定均已发展得比较成熟，但对于更远的复杂亲缘关系，如二级、三级、四级亲缘关系，仍然缺少有效的解决方案。因为两个个体之间的亲缘关系越远，所共享的等位基因越少，需要检测的遗传标记就越多，鉴定难度也越大。现有检测方法系统效能的不足和生物检材的降解是导致复杂亲缘关系推断困难的主要原因。

2、近年来，单核苷酸多态性(single nucleotide polymorphism，snp)成为备受关注的新型短片段遗传标记，在亲缘关系分析领域展现出了巨大潜力。比起传统的str遗传标记，它们的扩增片段短，在降解检材分析中具有独特优势；突变率远低于str，约为10-8；不受滑脱峰的干扰，更易于分型分析。虽然单个二等位遗传标记的鉴定效力较低，但在人类基因组中大约包括1500万个snp，可用的snp数量非常庞大，通过复合扩增和高通量测序技术，增加检测基因座的数量，同样可达到较高的亲缘鉴定效力。基于高通量测序平台，构建多重复合扩增系统，能够同时检测大量str和snp短片段遗传标记，增强对亲缘关系的鉴定效力。

3、目前，市场上身份识别效力最强的美国商品化检测试剂盒forenseq dnasignature prep kit只能满足部分二级亲缘关系鉴定，但对于三级和四级复杂亲缘关系鉴定，远远无法满足需求。

4、有鉴于此，特提出本发明。

技术实现思路

1、本发明的目的之一在于提供一种用于复杂亲缘关系分析的分子标记组合，以至少解决现有技术中存在的技术问题之一。

2、本发明的目的之二在于提供用于扩增上述分子标记组合的引物组。

3、本发明的目的之三在于提供包含上述引物组的试剂盒。

4、本发明的目的之四在于提供一种用于复杂亲缘关系鉴定的分析方法。

5、根据本发明的第一个方面，提供了一种用于复杂亲缘关系分析的分子标记组合，包括以下部分或全部snp位点：

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7、需要指出的是，以上2136个snp位点是在美国国立生物技术信息中心(nationalcenter for biotechnology information,ncbi)数据库中自主筛选得到的鉴定效力较高、满足身份识别的snp。该2136个snp位点是根据ncbi的snp数据库的命名方式来表示的。本领域技术人员知晓的其他表示方式，如以hgvs命名法标注某位点在参考gdna上的位置来表示。采用其他命名方式来标注指代与本发明一样的snp位点或snp位点组合也属于本发明的范围。

8、可以理解的是，本发明提供了2136个可用于复杂亲缘关系分析的snp位点，当进行亲缘关系鉴定时，可以选择上述2136个snp位点中的部分，例如10个、100个、1000个、1500个或2000个用于鉴定测序，也可以使用上述全部2136个snp位点用于鉴定测序。使用的snp位点越多，对复杂亲缘关系的鉴定结果越准确，优选使用上述全部2136个snp位点用于鉴定测序。

9、根据本发明的第二个方面，提供了一种用于复杂亲缘关系分析的引物组，包括用于扩增上述的部分或全部snp位点的引物组。

10、可以理解的是，本发明中所述的用于扩增上述的部分或全部snp位点的引物组指的是引物组与所使用的snp位点的对应关系，例如，当使用上述全部2136个snp位点用于鉴定测序时，则使用用于扩增上述全部snp位点的引物组。

11、进一步地，所述引物组包括如下表所示全部seq id no.1～seq id no.4276所示核苷酸序列的引物，共2138对，每对引物由正反向引物组成(例如p1中正向引物对应seq idno.1，反向引物对应seq id no.2，p2中正向引物对应seq id no.3，反向引物对应seq idno.4，p3中正向引物对应seq id no.5，反向引物对应seq id no.6等，以此类推)。为保证扩增效率的均衡性，一个扩增片段可能由1-2对引物进行扩增。

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57、可以理解的是，根据实际检测需要，可选用不同引物比例进行混合检测，本发明对此不做限定。

58、根据本发明的第三个方面，提供了一种用于复杂亲缘关系分析的试剂盒，包括上述的引物组。

59、可以理解的是，本发明提供的试剂盒除了包含上述引物组外，还可以包含进行pcr扩增实验时能够用到的其他试剂，例如dna聚合酶和缓冲液等，优选为缓冲液nb、缓冲液m、gdna、em808 polymerase mixture或ddh2o等。

60、根据本发明的第四个方面，提供了上述的用于亲缘关系分析的分子标记组合、引物组或试剂盒在在鉴定人类亲缘关系中的应用。

61、根据本发明的第五个方面，提供了一种亲缘关系的分析方法，所述方法包括：采用上述的引物组或试剂盒对待检测样本的dna进行扩增，然后对扩增产物进行测序文库构建并进行测序，以确定所述待检测样本中引物的覆盖位点的序列信息。

62、具体地，所述方法包括如下步骤：

63、a.获取待检测样本的dna；

64、b.靶向多重复合扩增dna，进行聚合酶pcr-1反应，并进行纯化；

65、c.以步骤b所得产物为模板，进行通用聚合酶pcr-2反应；

66、d.以步骤c所得产物为模板，进行dna纯化；

67、e.高通量测序；

68、d.数据分析，鉴定亲缘关系。

69、进一步地，所述扩增是所述引物组中部分或全部引物在同一反应体系中的复合扩增；

70、优选地，所述引物组合中各引物是一定比例进行混合。

71、进一步地，所述测序文库构建包括对扩增产物进行接头连接得到多重pcr文库。

72、进一步地，还包括对扩增产物和/或多重pcr文库进行纯化的步骤。

73、进一步地，在数据分析过程中，对测序文库进行质控，优选采用agilent 2100生物分析仪和high sensitivity dna chip进行质控。

74、进一步地，高通量测序可采用高通量测序仪，优选mgiseq2000测序平台。

75、进一步地，所述待检测样本的dna的输入量为1ng～5ng。

76、与现有技术相比，本发明具有如下有益效果：

77、本发明提供的用于复杂亲缘关系分析的分子标记组合，包含大量的snp位点，覆盖度广，适用对不同人群的分析与鉴定；通过千人基因组数据进行挖掘筛选获得的2136个snp位点，设计包括2136个遗传标记的复合扩增引物组，对个体身份鉴定的效力比现有商品化试剂盒更强，技术更加可靠。

78、本发明供的引物组，相互之间不会相互干扰，利用纯化后的dna作模板，能够实现对上述2136个遗传标记的同时扩增，通过一管反应即可获得包含2136个遗传标记的扩增产物，方便高效。

79、本发明通过自主筛选的2136个snp遗传标记，构建全新的、鉴定效力更强的多重复合扩增系统，用于四级内复杂亲缘关系鉴定，为降解检材的复杂亲缘关系鉴定提供进一步保障。