CN116926208A - 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 - Google Patents

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 Download PDF

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CN116926208A
CN116926208A CN202310998351.5A CN202310998351A CN116926208A CN 116926208 A CN116926208 A CN 116926208A CN 202310998351 A CN202310998351 A CN 202310998351A CN 116926208 A CN116926208 A CN 116926208A
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analysis
primer set
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complex genetic
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王升启
张庆珍
杨森
周喆
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

本发明提供了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，涉及个体身份识别技术领域。本发明提供的用于亲缘关系分析的分子标记组合，包含大量的SNP位点，广泛分布于人类基因组上，适用对不同群体的亲缘分析与鉴定。通过对千人基因组数据进行挖掘，筛选获得2136个个体识别效力较高的SNP位点，基于高通量测序平台，自主设计多重复合扩增引物组，对复杂亲缘关系的区分效力和准确率比现有商品化试剂盒更强，技术更加可靠。

Description

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
技术领域
本发明涉及个体身份识别技术领域，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
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。
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。
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。
有鉴于此，特提出本发明。
发明内容
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。
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。
本发明的目的之三在于提供包含上述引物组的试剂盒。
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。
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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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。
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。
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。
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。
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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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。
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。
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。
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。
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。
具体地，所述方法包括如下步骤：
a.获取待检测样本的DNA；
b.靶向多重复合扩增DNA，进行聚合酶PCR-1反应，并进行纯化；
c.以步骤b所得产物为模板，进行通用聚合酶PCR-2反应；
d.以步骤c所得产物为模板，进行DNA纯化；
e.高通量测序；
d.数据分析，鉴定亲缘关系。
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；
优选地，所述引物组合中各引物是一定比例进行混合。
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。
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。
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。
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。
进一步地，所述待检测样本的DNA的输入量为1ng～5ng。
与现有技术相比，本发明具有如下有益效果：
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。
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。
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。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的试剂盒对一级亲缘关系区分中的对数似然率分布图；
图2为本发明实施例提供的试剂盒对二级亲缘关系区分中的对数似然率分布图；
图3为本发明实施例提供的试剂盒对三级亲缘关系区分中的对数似然率分布图；
图4为本发明实施例提供的试剂盒对四级亲缘关系区分中的对数似然率分布图。
具体实施方式
除非本文另有定义，连同本发明使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。术语的含义和范围应当清晰，然而，在任何潜在不明确性的情况下，本文提供的定义优先于任何字典或外来定义。在本申请中，除非另有说明，“或”的使用意味着“和/或”。此外，术语“包括”及其他形式的使用是非限制性的。
一般地，连同本文描述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学以及蛋白和核酸化学和杂交使用的命名法和其技术是本领域众所周知和通常使用的那些。除非另有说明，本发明的方法和技术一般根据本领域众所周知，且如各种一般和更具体的参考文献中所述的常规方法来进行，所述参考文献在本说明书自始至终引用和讨论。酶促反应和纯化技术根据制造商的说明书、如本领域通常实现的或如本文所述来进行。连同本文描述的分析化学、合成有机化学以及医学和药物化学使用的命名法、以及其实验室程序和技术是本领域众所周知和通常使用的那些。
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。如无特别说明，实施例中的材料为根据现有方法制备而得，或直接从市场上购得。
本实施例中用到的主要仪器与试剂如下：
实施例1
一、样本
101例家系样本，包括64对全同胞关系(一级亲缘关系)、44对叔侄关系(二级亲缘关系)、97对第一代堂表兄(三级亲缘关系)、55对第二代堂表兄关系(四级亲缘关系)和2015对无关个体对。
二、实验方法
1.遗传标记筛选及引物设计
SNP筛选的对象为千人基因组项目收集的数据，包括全球26个不同群体2504个人的基因组数据。原始数据中含有多种变异类型，包括SNP、Indel，以及microhaplotype，总的位点数量约为84M。通过10项逐层筛选，最后有2218个位点被保留下来，筛选流程和参数设置如下：
(1)次等位基因频率，即在所有单倍型中第二大的等位基因频率。筛选过程中，次等位基因频率MAF的大小被设置为0.3，如果某一位点的次等位基因频率小于该值，则会被过滤掉。经过次等位基因频率的过滤，有2604207个位点被保留。
(2)处于非编码区。通过GENCODE确定了约1.87M个基因所对应的编码区，然后过滤掉所有处于编码区的位点，经过该项筛选，有1090037个位点被保留。
(3)远离常用STR区域，位点与其所在染色体上的给定STR的距离要大于5Mbp。筛选过程中，一共使用了67个分布在所有染色体上的STR。经过该项筛选，有850061个位点被保留。
(4)Hardy-Weinberg平衡检验。当位点平衡检验的结果小于给定的P值时被过滤掉，P值被设置为0.05。经过该项筛选，有180968个位点被保留。
(5)期望杂合度。当某一位点的期望杂合度小于0.45时即被过滤掉。经过该项筛选，有138967个位点被保留。
(6)非Indel变异，即过滤掉是Indel变异的位点。经过该项筛选，有115556个位点被保留。
(7)种群分化程度。当一个位点的种群分化系数大于0.03时被过滤掉。经过该项筛选，有51297个位点被保留。
(8)与特定SNP位点是否存在连锁不平衡。位点与其所在染色体上的给定SNP的连锁不平衡系数超过0.01时即被过滤。筛选过程中，一共使用了94个分布在所有常染色体上的位点。经过该项筛选，有50759个位点被保留。
(9)根据位点距离降采样，即同一染色体上的任意两个位点之间的距离需要大于100Kbp。经过该项降采样，有2543个位点被保留。
(10)根据连锁不平衡降采样，即同一染色体上的任意两个位点之间的连锁不平衡系数需要大于0.01，经过该项降采样，有2219个SNP位点被保留。
(11)设计多重扩增引物，2136对SNP设计成功。
根据SNP位点设计引物序列如P1～P2138所示(对应SEQ ID NO.1～SEQ IDNO.4276，例如P1中正向引物对应SEQ ID NO.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 ID NO.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)。
2.准备样本DNA
本发明对DNA的输入量存在要求。扩增特定基因或区域所需新制DNA量为1-5ng。
3.文库构建
(1)多重PCR-1靶向扩增。根据样本数量，按照说明书配制PCR反应体系：
PCR扩增程序为：
95℃，3min 30s；
30个循环：98℃，20s；60℃，10min；
72℃，5min；保持在4℃。
(2)磁珠纯化产物。30μl反应体系中加入1.2倍体积的磁珠(36μl)，吸打或涡旋混匀，室温静置5min。向管内加入50μl YF缓冲液B，吸打混匀，室温静置5min。小心移弃上清，向PCR管内加入180μl 80％乙醇溶液静置30s。移弃上清，再次向PCR管内加入180μl 80％乙醇溶液，静置30s移弃上清。小心使用10μl移液器移弃底部残留的乙醇。保持PCR管在磁力架上，室温静置3～5min，晾干磁珠，使残留的乙醇彻底挥发。加入24μl Nuclease FreeWater，将PCR管从磁力架上取下，吸打或涡旋混匀，室温静置2min。瞬时离心，将PCR管置于磁力架上2min，待溶液澄清。用移液器吸取13.5μl上清液，转移到新的PCR管中，管内上清液为合并后的多重PCR产物。
(3)PCR-2接头连接反应。配制PCR体系：
PCR扩增程序为：
95℃，3min 30s；
9个循环：98℃，20s；58℃，1min；72℃，30s；
72℃，5min；保持在4℃。
(4)文库纯化。提前取出磁珠，充分涡旋振荡，室温放置至少30min。30μl反应体系中加入1.2倍体积的磁珠(36ul)，吸打或涡旋混匀室温静置5min。瞬时离心，将PCR管置于磁力架上3min，待溶液澄清，彻底移除上清，将PCR管从磁力架取下，向管内加入50μl YF缓冲液B，吸打混匀，室温静置5min。瞬时离心，将PCR管置于磁力架上3min。保持PCR管在磁力架上，小心移弃上清，向PCR管内加入180μl 80％乙醇溶液静置30s。保持PCR管在磁力架上移弃上清，再次向PCR管内加入180μl 80％乙醇溶液，静置30s移弃上清。盖上管盖，瞬时离心，将残留乙醇离心至管底，将PCR管置于磁力架上，小心使用10μl移液器移弃底部残留的乙醇，注意不要吸到磁珠。保持PCR管在磁力架上，室温静置3～5min，晾干磁珠，使残留的乙醇彻底挥发。加入24μl Nuclease Free Water，将PCR管从磁力架上取下，吸打或涡旋混匀，室温静置2min。瞬时离心，将PCR管置于磁力架上2min，待溶液澄清。用移液器吸取20μl上清液，转移到新的EP管中，管内上清液为制备好的多重PCR文库。
4.DNA文库质控
采用DNA高灵敏芯片(High Sensitivity DNA Chip)及Agilent 2100生物分析仪对DNA文库进行质量控制，方法如下：
(1)准备胶和荧光混合液。从冰箱中取出胶和荧光试剂，室温放置30min。取15μl荧光溶液加到胶内，振荡混匀，离心后，将混合液转移至过滤柱中进行离心，离心力设置为2240g±20％，离心时间为10min。离心结束后，弃去过滤柱。室温避光保存。
(2)取出芯片置于桌面，加入9μl胶至标有黑色“G”的孔中，使用注射器将胶压下1min，松开开关，待注射器回复到原来位置；
(3)在其余3个标有“G”的孔中各加入9μl胶；
(4)在剩余每个样本孔内加5μl内标(marker)溶液；
(5)在标有梯子形状的孔内加1μl Ladder，在剩余11个样本孔内加1μl样本，置于IKA振荡器上以2400rpm振荡1min；
(6)将芯片置于Agilent 2100生物分析仪上，选择芯片类型，对样本进行命名，运行程序，开始检测，观察产生的峰图，判断建库质量。
5.双barcode环化文库构建
采用MGIEasy双barcode环化试剂盒，方法如下：
(1)变性。根据Input DNA的片段长度，取1pmol DNA至0.2ml PCR管中，用TEBuffer补充至总体积48μl。将PCR管至于PCR仪上，变性条件为：热盖105℃；95℃，3min。反应结束后，立即将PCR管转移至冰上，放置2min。
(2)单链环化。在冰上配制单链环化反应液：
将12.1μl单链环化反应液加入反应后的PCR管中，涡旋震荡6次，每次3s，瞬时离心将反应液收集至管底。将PCR管置于PCR仪上，按照热盖105℃，37℃，30min；4℃保持。反应结束后，取出PCR管，立即进入下步反应。
(3)酶切消化。在冰上配制酶切消化反应液：
将上述4μl酶切消化反应液加入反应后的PCR管中，涡旋震荡6次，每次3s，瞬时离心将反应液收集至管底。将PCR管置于PCR仪上，按照热盖105℃，37℃30min；4℃保持。反应结束后，向每个反应中加入7.5μl Digestion Stop Buffer，涡旋震荡6次，每次3s，瞬时离心将反应液收集至管底。所有反应液转移到新的1.5ml EP管中。
(4)环化产物纯化。提前30min取出DNA Clean Beads置于室温，使用前充分震荡混匀。吸取170μl DNA Clean Beads至产物中，用移液器轻轻吹打至少10次至完全混匀，最后一次应确保将吸头中所有液体及磁珠都打入1.5ml EP管中。室温孵育10min。瞬时离心，将1.5ml EP管置于磁力架，静置2-5min至液体澄清，用移液器小心吸取并丢弃上清。保持1.5ml EP管置于磁力架上，加入500μl新鲜配制的80％乙醇漂洗磁珠及管壁，小心吸取并丢弃上清。重复上一步，尽量吸干管内液体。保持1.5ml EP管固定于磁力架上，打开1.5ml EP管管盖，室温干燥，直至磁珠表面无反光、无开裂。将1.5ml EP管从磁力架上取下，加入22μlTE Buffer进行DNA洗脱，用移液器轻轻吹打至少10次至完全混匀。室温下溶解10min。瞬时离心，将1.5ml EP管置于磁力架上，静置2-5min至液体澄清，将20μl上清液转移到新的1.5ml EP管中。使用ssDNA Assay Kit单链DNA荧光定量试剂盒，按照定量试剂盒的操作说明对环化纯化后产物进行定量。
6.DNB制备
(1)取出文库、DNB制备缓冲液、DNB聚合酶混合液、TE缓冲液和DNB终止缓冲液，置于冰盒上约0.5h。待融化后，使用漩涡振荡器振荡混匀5s后，短暂离心置于冰盒上备用。
(2)取PCR管，在冰上按如下体系配制反应混合液：
注：文库投入体积V(μl)＝N*330g/mol*60fmol/(1000*1000*C)
N为核苷酸数目(单位nt，文库总片段长度)，C为文库的浓度ng/μl
(3)将反应体系混匀并瞬时离心，置于PCR仪中进行引物杂交，反应条件为：热盖105℃，95℃，1min；65℃，1min；40℃，1min；4℃保持。
(4)取出DNB聚合酶混合液Ⅱ(LC)短暂离心5s后置于冰盒上备用。
(5)PCR仪达到4℃后取出PCR管，短暂离心5s后，在冰上加入40μl的DNB聚合酶混合液和4μl的DNB聚合酶混合液Ⅱ(LC)。
(6)反应混合液用漩涡振荡器振荡混匀，迷你离心机离心5s，置于PCR仪中反应，反应条件如下：热盖35℃，30℃25min，4℃保持。
(7)PCR仪到4℃后立即加入20μl DNB终止缓冲液，用阔口吸头缓慢吸打混匀5-8次。
(8)DNB浓度测定。DNB浓度8ng/μl以上为合格，浓度不合格的需重新制备；如浓度超过40ng/μl，需要用DNB加载缓冲液I稀释至20ng/μl后使用。
7.DNB加载
(1)取出新的EP管，按下表所示配制DNB加载体系：
(2)DNB加载体系用阔口吸头缓慢混匀5-8次。切勿离心、震荡及剧烈吹打。每条lane需要30μl DNB加载体系。
(3)将密封垫放置在密封垫槽中，将载片放置在加样器中。用移液器吸取30μl混匀后DNB样品，将阔口吸头插入流路入口中，不要按下移液器的控制按钮。按下移液器上的吸头脱卸按钮，样本会自动流入芯片中。
(4)确保样本加载完成后，逆时针旋转拔出吸头。将加样器正面朝上，水平放置30分钟，即刻转移到测序仪上使用。
8.高通量测序
(1)准备SE400测序试剂盒。取出测序试剂槽，待融化后，颠倒混匀3次。取出dNTPs混合液和dNTPs混合液II，室温融化后置于冰盒上备用，然后取出测序酶混合液。打开试剂槽盖板，用无尘纸擦干冷凝水。用1ml吸头在1号和2号孔边缘分别戳出一个直径小于1cm的孔。根据不同试剂盒，将试剂加入1和2号孔。
(2)装载测序试剂槽。取出清洗试剂槽，使用无尘纸擦拭试剂仓底部的冷凝水，保持干燥和干净。按照测序试剂槽盖板指示方向，把准备好的测序试剂槽轻轻推进试剂仓，直到推到底部并确认完全放入。用扫码枪扫描测序试剂盒条码，关闭试剂仓门，点击“下一步”。
(3)装载载片。打开载片仓门，一手压住水洗载片两侧，一手按下载片吸附按钮，待真空释放后，将清洗载片从平台上取出。取出载片，用扫码枪将载片ID输入到文本框中。按下载片吸附按钮。载片标签呈右上放置，双手握住载片两侧，载片定位孔对准定位柱放置，同时向左上角45°轻轻推动，保持载片孔内壁与定位柱贴合，然后同时按下载片两侧边框，使载片吸附在平台上。
(4)开始测序。填写各项测序参数，对填写的各项信息进行复核，确保准确无误。点击开始，仪器开始测序，测序结束后要进行仪器清洗。
9.结果分析
(1)二代测序数据分析
采用CLC workbench 22.0software分析SNP数据，测序深度阈值设置为30×，最低检出频率设置为20％，其余参数采用软件默认值。(2)不同类型的复杂亲缘关系鉴定效力分析
一级到四级内复杂亲缘关系的对数似然率频率分布直方图如下图1-4所示，灵敏度、特异性及准确率如下表所示。当log10LR阈值取传统的2和-2时，一级、二级亲缘关系鉴定的灵敏度、特异性、准确率均为100％，三级亲缘关系鉴定的灵敏度、特异性、准确率分别为100％、99.55％和99.57％，四级亲缘关系鉴定灵敏度、特异性、准确率分别为100％、96.23％和96.33％。比起现有Illumina公司的商品化法医高通量测序试剂盒ForenSeq DNASignature Prep Kit，本试剂盒具有更强的亲缘鉴定效力，对二级亲缘鉴定的系统效能提高17.51％，三级亲缘关系鉴定的系统效能提高59.08％，并能够为部分四级亲缘关系鉴定提供有效解决方案。
表四级内复杂亲缘关系鉴定效力分析
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

1.一种用于复杂亲缘关系分析的分子标记组合，其特征在于，包括以下部分或全部SNP位点：
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2.一种用于复杂亲缘关系分析的引物组，其特征在于，包括用于扩增权利要求1所述的部分或全部SNP位点的引物组。
3.根据权利要求2所述的引物组，其特征在于，所述引物组包括部分或全部SEQ IDNO.1～SEQ ID NO.4276所示核苷酸序列的引物。
4.一种用于复杂亲缘关系分析的试剂盒，其特征在于，包括权利要求2或3所述的引物组。
5.权利要求1所述的用于复杂亲缘关系分析的分子标记组合、权利要求2或3所述的引物组或权利要求4所述的试剂盒在在鉴定人类亲缘关系中的应用。
6.一种亲缘关系鉴定的分析方法，其特征在于，所述方法包括：采用权利要求2或3所述的引物组、或权利要求4所述的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。
7.根据权利要求6所述的分析方法，其特征在于，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。
8.根据权利要求7所述的分析方法，其特征在于，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。
9.根据权利要求8所述的分析方法，其特征在于，还包括对扩增产物和/或多重PCR文库进行纯化的步骤。
10.根据权利要求6-9任一项所述的分析方法，其特征在于，所述待检测样本的DNA的输入量为1ng～5ng。
CN202310998351.5A 2023-08-09 2023-08-09 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 Pending CN116926208A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117524308A (zh) * 2023-05-23 2024-02-06 公安部鉴定中心 用于推测人亲缘关系等级的snp位点组合及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117524308A (zh) * 2023-05-23 2024-02-06 公安部鉴定中心 用于推测人亲缘关系等级的snp位点组合及其应用

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