CN105695575A - 55个snp位点多色荧光复合检测的方法及试剂盒 - Google Patents
55个snp位点多色荧光复合检测的方法及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种55个SNP位点多色荧光复合检测的方法及试剂盒,涉及包括核酸的测定或检验方法技术领域。试剂盒包括PCR引物,DNA聚合酶,PCR缓冲液,核酸外切酶Ⅰ,虾碱性磷酸酶,延伸引物,SNaPshot反应混合液,Hi-Di甲酰胺和GeneScanTM Size Standards-120 LIZ;PCR引物和延伸引物由55个SNP基因位点的引物组成,分别针对包含55个SNP位点的DNA片段。采用本发明可以快速、准确地获得55个SNP位点的基因分型,并通过荧光自动分型设备检测分析,对人类生物检材进行个体识别,具有很大优势和潜力,具有很高的***效能。
Description
技术领域
本发明涉及包括核酸的测定或检验方法技术领域。
背景技术
单核苷酸多态性(SingleNucleotidePolymorphisms,SNPs)是指人类基因组中特定位置核苷酸单个碱基序列的变异,一般是二等位基因,且其中最少的一种在人群中的频率不少于1%。与限制性片段长度多态性(RestrictionFragmentLengthPolymorphism,RELP)和短串联重复序列(ShortTandemRepeats,STRs)相比,第三代遗传标记SNPs分布更为广泛,占基因组已知多态性的80%,是最常见的遗传变异类型。近年来,SNPs在法医学领域受到越来越多的关注,主要由于其具有以下三方面的优点:(1)SNPs是检测单碱基变异,可以通过设计较短的扩增片段扩增小于100bp的DNA模板,弥补STRs在分析降解检材方面的局限性;(2)SNPs的突变率(10-8)比STR(10-3)低,这使其在亲缘关系鉴定方面更具价值;(3)二等位基因的特性使其检测方法多样,并且能够实现快速自动化分型;(4)利用SNPs能够进行祖先信息以及表型信息的推断,推测嫌疑人或失踪人员特征,从而缩小侦查范围,为侦破案件提供线索。SNPs最大的缺点是其多态性差,需要检测更多的SNP位点才能达到较高的识别率。而对于法医学检验中经常面对的有限的检材,一个稳定的、能同时检测多个位点的方法是十分有价值的,高效、省时且经济。
发明内容
本发明要解决的技术问题是提供一种用于个体识别的55个SNP位点多色荧光复合检测的方法及试剂盒,在专利ZL201010265870.3原有44个位点的基础上,将其扩展为55个位点的复合分型体系,提高方法的检验效能;优化反应条件,使检测方法更加稳定和经济;增加性别检测位点,使检测方法能够鉴别检材来源的性别。
为解决上述技术问题,本发明所采取的技术方案是:55个SNP位点多色荧光复合检测的试剂盒,其包括a)扩增体系:PCR缓冲溶液、MgCl2、dNTPs、PCR引物、TaqDNA聚合酶、模板DNA,b)扩增产物纯化试剂:核酸外切酶Ⅰ、虾碱性磷酸酶,c)延伸反应试剂:延伸引物、SNaPshot反应混合液,d)延伸产物纯化试剂:虾碱性磷酸酶,e)测试试剂:Hi-Di甲酰胺和GeneScanTMSizeStandards-120LIZ,PCR引物和延伸引物由55个SNP基因位点的引物组成,所述55个SNP基因位点在NCBI中SNP数据库的rs号分别为rs1109037,rs3780962,rs6591147,rs2342747,rs9951171,rs13182883,rs10092491,rs6955448,rs6444724,rs430046,rs321198,rs159606,rs560681,rs214955,rs13218440,rs9905977,rs1821380,rs279844,rs8078417,rs4606077,rs445251,rs987640,rs4530059,rs338882,rs10488710,rs6811238,rs722290,rs7041158,rs7520386,rs9866013,rs1498553,rs2270529,rs1478829,rs1523537,rs4364205,rs985492,rs12997453,rs740598,rs1736442,rs1294331,rs993934,rs10776839,rs315791,rs576261,rs2399332,rs2272998,rs7205345,rs2269355,rs1058083,rs10773760,rs464663,rs221956,rs1336071,rs1027895和位于人类牙釉质蛋白基因Amelogeningene上的一个性别检测SNP位点,所述性别检测SNP位点在女性个体中为纯合子T/T,在男性个体中为杂合子C/T,性别检测SNP位点的5’侧翼序列为TTTTACCTTCTTCTTTCT,3’侧翼序列为TTGTAGAACTCACATTCTCAGGC。
除性别检测SNP位点外,其余54个SNP基因位点根据以下标准进行选取:(1)具有高的杂合度(≥0.4);(2)等位基因频率在全球各大人群之间差异小(Fst<0.06);(3)***内SNPs之间要处于连锁平衡状态,位点之间的物理距离至少大于1Mb;(4)***内的SNP数目要足够多以达到个体识别效能;所有SNP位点的群体遗传学数据已经在公开刊物上发表。其相关信息见表1。
表1-续1
表1-续2
55个SNP基因位点的PCR引物和延伸引物的序列为:
rs1109037,PCR引物SEQIDNO.1-2,延伸引物SEQIDNO.111;
rs3780962,PCR引物SEQIDNO.3-4,延伸引物SEQIDNO.112;
rs6591147,PCR引物SEQIDNO.5-6,延伸引物SEQIDNO.113;
rs2342747,PCR引物SEQIDNO.7-8,延伸引物SEQIDNO.114;
rs9951171,PCR引物SEQIDNO.9-10,延伸引物SEQIDNO.115;
rs13182883,PCR引物SEQIDNO.11-12,延伸引物SEQIDNO.116;
rs10092491,PCR引物SEQIDNO.13-14,延伸引物SEQIDNO.117;
rs6955448,PCR引物SEQIDNO.15-16,延伸引物SEQIDNO.118;
rs6444724,PCR引物SEQIDNO.17-18,延伸引物SEQIDNO.119;
rs430046,PCR引物SEQIDNO.19-20,延伸引物SEQIDNO.120;
rs321198,PCR引物SEQIDNO.21-22,延伸引物SEQIDNO.121;
rs159606,PCR引物SEQIDNO.23-24,延伸引物SEQIDNO.122;
rs560681,PCR引物SEQIDNO.25-26,延伸引物SEQIDNO.123;
rs214955,PCR引物SEQIDNO.27-28,延伸引物SEQIDNO.124;
rs13218440,PCR引物SEQIDNO.29-30,延伸引物SEQIDNO.125;
rs9905977,PCR引物SEQIDNO.31-32,延伸引物SEQIDNO.126;
rs1821380,PCR引物SEQIDNO.33-34,延伸引物SEQIDNO.127;
rs279844,PCR引物SEQIDNO.35-36,延伸引物SEQIDNO.128;
rs8078417,PCR引物SEQIDNO.37-38,延伸引物SEQIDNO.129;
rs4606077,PCR引物SEQIDNO.39-40,延伸引物SEQIDNO.130;
rs445251,PCR引物SEQIDNO.41-42,延伸引物SEQIDNO.131;
rs987640,PCR引物SEQIDNO.43-44,延伸引物SEQIDNO.132;
rs4530059,PCR引物SEQIDNO.45-46,延伸引物SEQIDNO.133;
rs338882,PCR引物SEQIDNO.47-48,延伸引物SEQIDNO.134;
rs10488710,PCR引物SEQIDNO.49-50,延伸引物SEQIDNO.135;
rs6811238,PCR引物SEQIDNO.51-52,延伸引物SEQIDNO.136;
rs722290,PCR引物SEQIDNO.53-54,延伸引物SEQIDNO.137;
rs7041158,PCR引物SEQIDNO.55-56,延伸引物SEQIDNO.138;
rs7520386,PCR引物SEQIDNO.57-58,延伸引物SEQIDNO.139;
rs9866013,PCR引物SEQIDNO.59-60,延伸引物SEQIDNO.140;
rs1498553,PCR引物SEQIDNO.61-62,延伸引物SEQIDNO.141;
rs2270529,PCR引物SEQIDNO.63-64,延伸引物SEQIDNO.142;
rs1478829,PCR引物SEQIDNO.65-66,延伸引物SEQIDNO.143;
rs1523537,PCR引物SEQIDNO.67-68,延伸引物SEQIDNO.144;
rs4364205,PCR引物SEQIDNO.69-70,延伸引物SEQIDNO.145;
rs985492,PCR引物SEQIDNO.71-72,延伸引物SEQIDNO.146;
rs12997453,PCR引物SEQIDNO.73-74,延伸引物SEQIDNO.147;
rs740598,PCR引物SEQIDNO.75-76,延伸引物SEQIDNO.148;
rs1736442,PCR引物SEQIDNO.77-78,延伸引物SEQIDNO.149;
rs1294331,PCR引物SEQIDNO.79-80,延伸引物SEQIDNO.150;
rs993934,PCR引物SEQIDNO.81-82,延伸引物SEQIDNO.151;
rs10776839,PCR引物SEQIDNO.83-84,延伸引物SEQIDNO.152;
rs315791,PCR引物SEQIDNO.85-86,延伸引物SEQIDNO.153;
rs576261,PCR引物SEQIDNO.87-88,延伸引物SEQIDNO.154;
rs2399332,PCR引物SEQIDNO.89-90,延伸引物SEQIDNO.155;
rs2272998,PCR引物SEQIDNO.91-92,延伸引物SEQIDNO.156;
rs7205345,PCR引物SEQIDNO.93-94,延伸引物SEQIDNO.157;
rs2269355,PCR引物SEQIDNO.95-96,延伸引物SEQIDNO.158;
rs1058083,PCR引物SEQIDNO.97-98,延伸引物SEQIDNO.159;
rs10773760,PCR引物SEQIDNO.99-100,延伸引物SEQIDNO.160;
rs464663,PCR引物SEQIDNO.101-102,延伸引物SEQIDNO.161;
rs221956,PCR引物SEQIDNO.103-104,延伸引物SEQIDNO.162;
rs1336071,PCR引物SEQIDNO.105-106,延伸引物SEQIDNO.163;
rs1027895,PCR引物SEQIDNO.107-108,延伸引物SEQIDNO.164;
位于人类牙釉质蛋白基因Amelogeningene上的一个性别检测SNP位点,PCR引物SEQIDNO.109-110,延伸引物SEQIDNO.165。
55个SNP基因位点的PCR引物和延伸引物的序列也可参见表2。本发明是对专利“44个SNP位点多色荧光复核检测的方法及试剂盒”(专利号ZL201010265870.3)的改进及发展。与专利ZL201010265870.3相比,去掉了一个位点rs4288409,增加了12个位点,其rs号分别为rs6591147,rs6444724,rs159606,rs4606077,rs9866013,rs2270529,rs1478829,rs4364205,rs985492,rs464663,rs1027895和一个性别检测SNP位点。根据NCBI的SNP数据库(dbSNP)提供的DNA序列,设计了新增12个位点的扩增引物及延伸引物;原有44个SNP位点多色荧光复合检测的方法中4个位点的扩增引物被重新设计,分别为rs13218440,rs8078417,rs7520386和rs1336071,其中rs13218440重新设计了上游引物,其余3个位点重新设计了上下游引物;28条延伸引物被修改,其中5条更改了引物结合方向,分别为rs279844,rs7041158,rs1498553,rs221956和rs1336071,23条更改了引物长度,分别为rs2342747,rs9951171,rs13182883,rs10092491,rs6955448,rs13218440,rs9905977,rs987640,rs338882,rs10488710,rs6811238,rs722290,rs7520386,rs1523537,rs993934,rs10776839,rs315791,rs576261,rs2272998,rs7205345,rs2269355,rs1058083和rs10773760。55个SNP位点的扩增产物长度为56-125bp,延伸引物长度为19-78bp。
表2-续1
表2-续2
表2-续3
利用55个SNP位点多色荧光复合检测的试剂盒进行个体识别的测试方法,按照下列步骤进行:
a)提取DNA,得DNA提取液;
b)在扩增体系中加入步骤a)所得的DNA提取液,然后在扩增仪中进行扩增,扩增条件为:95℃、5min预变性;然后依次在95℃、30s,60℃、30s,65℃、30s,进行35个循环;再在65℃保持7min;最终在4℃保存,获得扩增产物;
c)应用核酸外切酶Ⅰ和虾碱性磷酸酶纯化扩增产物,纯化反应条件:37℃、1h,然后80℃、15min,最终在4℃保存,得纯化后的扩增产物;
d)对纯化后的扩增产物进行延伸反应
将纯化后的扩增产物加入至所述的延伸反应试剂中进行延伸反应;所述的延伸反应的条件:依次在96℃、10s,58℃、5s,60℃、30s进行50个循环,最终保存在4℃,得延伸产物;
与专利ZL201010265870.3相比,循环条件中第二步温度由50℃提高到58℃,循环次数由25次增加至50次;
e)应用虾碱性磷酸酶纯化延伸产物
将虾碱性磷酸酶加入延伸产物中,在下列条件下进行纯化反应:37℃、1h,80℃、15min,最终保存在4℃,得纯化后的延伸产物;
与专利ZL201010265870.3相比,是将虾碱性磷酸酶加入延伸产物中,使全部延伸产物纯化,且纯化条件中第二步温度由65℃提高到80℃;
f)检测、确定产物,
将步骤e)中得到的纯化后的延伸产物中加入至所述的测试试剂中,混匀;然后95℃变性3min,4℃迅速冷却后进行电泳检测;最后根据延伸产物峰的位置和颜色确定55个SNP基因位点的基因型。
采用上述技术方案所产生的有益效果在于:采用本发明可以快速、准确地获得55个SNP位点的基因分型,并通过荧光自动分型设备检测分析,对人类生物检材进行个体识别。本发明在对高度降解检材DNA的法医学个体识别具有很大的优势和潜力;较专利ZL201010265870.3,将44个位点的复合分型体系增加到55个位点,具有更高的***效能。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明;
图1和图2是本发明对某一DNA样本的分型结果图。
具体实施方式
用于个体识别的55个SNP位点多色荧光复合检测的试剂盒,其包括a)扩增体系:PCR缓冲溶液、MgCl2、dNTPs、PCR引物、TaqDNA聚合酶、模板DNA,b)扩增产物纯化试剂:核酸外切酶Ⅰ、虾碱性磷酸酶,c)延伸反应试剂:延伸引物、SNaPshot反应混合液,d)延伸产物纯化试剂:虾碱性磷酸酶,e)测试试剂:Hi-Di甲酰胺和GeneScanTMSizeStandards-120LIZ,PCR引物和延伸引物由55个SNP基因位点的引物组成,55个SNP基因位点在NCBI中SNP数据库的rs号分别为rs1109037,rs3780962,rs6591147,rs2342747,rs9951171,rs13182883,rs10092491,rs6955448,rs6444724,rs430046,rs321198,rs159606,rs560681,rs214955,rs13218440,rs9905977,rs1821380,rs279844,rs8078417,rs4606077,rs445251,rs987640,rs4530059,rs338882,rs10488710,rs6811238,rs722290,rs7041158,rs7520386,rs9866013,rs1498553,rs2270529,rs1478829,rs1523537,rs4364205,rs985492,rs12997453,rs740598,rs1736442,rs1294331,rs993934,rs10776839,rs315791,rs576261,rs2399332,rs2272998,rs7205345,rs2269355,rs1058083,rs10773760,rs464663,rs221956,rs1336071,rs1027895和位于人类牙釉质蛋白基因Amelogeningene上的一个性别检测SNP位点,所述性别检测SNP位点在女性个体中为纯合子T/T,在男性个体中为杂合子C/T,性别检测SNP位点的5’侧翼序列为TTTTACCTTCTTCTTTCT,3’侧翼序列为TTGTAGAACTCACATTCTCAGGC。
55个SNP基因位点的PCR引物和延伸引物的序列、浓度分别参见表2和表3。
延伸引物包括延伸引物混合物(29个位点)和延伸引物混合物(26个位点),由不同长度的延伸引物组成,分别针对55个SNP位点的多态性位点。
表3-续1
表3-续2
利用上述的试剂盒进行个体识别的测试方法,按照下列步骤进行:
a)提取DNA,得DNA提取液;
b)多重PCR扩增包含55个SNP位点的DNA片段
在扩增体系中加入步骤a)所得的DNA提取液,然后在扩增仪中进行扩增。PCR扩增体系的组分、浓度或含量及与44个SNP复合分型体系的比较如下:
*试剂盒提取的DNA在体系中加入0.5-2ng,Chelex-100提取的DNA在体系中加入5-20ng
扩增的热循环参数:
得扩增产物;
c)应用核酸外切酶Ⅰ(ExoⅠ)和虾碱性磷酸酶(SAP)纯化步骤b)所得的扩增产物,纯化反应体系及与44个SNP复合分型体系的比较如下:
扩增产物纯化反应的条件:
得到纯化后的扩增产物;
d)将纯化后的扩增产物加入至所述的延伸反应试剂中进行延伸反应,延伸反应的组分、含量及与44个SNP复合分型体系的比较如下:
延伸反应的热循环参数及与44个SNP复合分型体系的比较如下:
得延伸产物;
e)应用虾碱性磷酸酶纯化延伸产物,纯化反应体系与44个SNP复合分型体系的比较如下:
纯化反应的热循环参数及与44个SNP复合分型体系的比较如下:
得纯化后的延伸产物;
f)检测、确定产物,
将步骤e)中得到的纯化后的延伸产物中加入Hi-Di甲酰胺和GeneScanTMSizeStandards-120LIZ混匀,体系配制及与44个SNP复合分型体系的比较如下:
然后95℃变性3min,4℃迅速冷却后,用美国AB公司的DNA自动分析仪进行电泳检测,检测条件:1500V电压,36cm毛细管,POP4凝胶,电泳20min;应用DataCollection3.0软件收集数据,GenemapperV3.2软件进行结果分析。其中某一DNA样本的分型结果图见图1和图2所示。图1即Plex1,包括29个SNP位点的分型结果,图2即Plex2,包括26个SNP的分型结果。每张图自上而下四个泳道分别表示检测到的G、A、C、T四种碱基。对于每一个SNP位点,其两个等位基因分别位于不同的泳道。荧光自动分型设备根据峰出现在哪一泳道以及在泳道中的位置自动得出55个位点的分型,即每个峰横坐标标记的结果。
SEQUENCELISTING
<110>河北医科大学
<120>55个SNP位点多色荧光复合检测的方法及试剂盒
<130>2016
<160>165
<170>PatentInversion3.3
<210>1
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<212>DNA
<213>人工序列
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ctggtggtgagggtggagatt21
<210>2
<211>21
<212>DNA
<213>人工序列
<400>2
gtgccagtgcgagatgaaagt21
<210>3
<211>23
<212>DNA
<213>人工序列
<400>3
ctggtggtgtgtattctgcggta23
<210>4
<211>23
<212>DNA
<213>人工序列
<400>4
aacatgggatgaacaaggtcaag23
<210>5
<211>25
<212>DNA
<213>人工序列
<400>5
tactctccataagatagccttccac25
<210>6
<211>23
<212>DNA
<213>人工序列
<400>6
ctggtctgctcagctaacatctc23
<210>7
<211>21
<212>DNA
<213>人工序列
<400>7
gtcagcatgggaggaagaaaa21
<210>8
<211>21
<212>DNA
<213>人工序列
<400>8
ggcagcagtaaccagcaacac21
<210>9
<211>21
<212>DNA
<213>人工序列
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agtcctcgttgttcctctggg21
<210>10
<211>21
<212>DNA
<213>人工序列
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ctgctttcatgctgggacctg21
<210>11
<211>21
<212>DNA
<213>人工序列
<400>11
ggggtcccttctggcctagta21
<210>12
<211>23
<212>DNA
<213>人工序列
<400>12
cgttactttcttcctgcctttgt23
<210>13
<211>24
<212>DNA
<213>人工序列
<400>13
agtggcattagaaattccagatag24
<210>14
<211>22
<212>DNA
<213>人工序列
<400>14
ccccgcaaactaactaggataa22
<210>15
<211>25
<212>DNA
<213>人工序列
<400>15
cctgccgttactactattgttgaaa25
<210>16
<211>28
<212>DNA
<213>人工序列
<400>16
cttgtggagttattcttatgtaatcgtc28
<210>17
<211>26
<212>DNA
<213>人工序列
<400>17
gaaaggactaaattgttgaacactgg26
<210>18
<211>25
<212>DNA
<213>人工序列
<400>18
tcctactccgtagtaaatgagagca25
<210>19
<211>25
<212>DNA
<213>人工序列
<400>19
gaacaaggtcatacaatgaatggtg25
<210>20
<211>25
<212>DNA
<213>人工序列
<400>20
cacctatgggctcttcttatttctc25
<210>21
<211>23
<212>DNA
<213>人工序列
<400>21
cctacacacaggcttcaggttac23
<210>22
<211>25
<212>DNA
<213>人工序列
<400>22
atgtgcgtttctccacactttatac25
<210>23
<211>30
<212>DNA
<213>人工序列
<400>23
gcacaaacatctaataagcactttcaatca30
<210>24
<211>29
<212>DNA
<213>人工序列
<400>24
cattggagagaataaaatataggagacag29
<210>25
<211>24
<212>DNA
<213>人工序列
<400>25
catctgttcaggtttctctccatc24
<210>26
<211>24
<212>DNA
<213>人工序列
<400>26
cacagcctcttactgcacttcata24
<210>27
<211>25
<212>DNA
<213>人工序列
<400>27
tcatcaactttatcgctttttcctg25
<210>28
<211>21
<212>DNA
<213>人工序列
<400>28
ctttattctggcagcccttgg21
<210>29
<211>24
<212>DNA
<213>人工序列
<400>29
gctgtggactgaaacttgatcctg24
<210>30
<211>23
<212>DNA
<213>人工序列
<400>30
aacccatcccagctgagtattcc23
<210>31
<211>23
<212>DNA
<213>人工序列
<400>31
aggaaaattcatgagctggtgtc23
<210>32
<211>20
<212>DNA
<213>人工序列
<400>32
ccaagcctgagggacaaagc20
<210>33
<211>22
<212>DNA
<213>人工序列
<400>33
cacaatggagccactgaactgc22
<210>34
<211>22
<212>DNA
<213>人工序列
<400>34
ttccttgtcccttctgacctta22
<210>35
<211>25
<212>DNA
<213>人工序列
<400>35
aatggcaacttctgataaaggataa25
<210>36
<211>29
<212>DNA
<213>人工序列
<400>36
aatatcaaggaagtttagagagttgtgag29
<210>37
<211>20
<212>DNA
<213>人工序列
<400>37
tgtacgtggtcaccagggga20
<210>38
<211>23
<212>DNA
<213>人工序列
<400>38
gcatctgagtgtgagaagagcct23
<210>39
<211>25
<212>DNA
<213>人工序列
<400>39
tgcctgtggctttgtagttctagtg25
<210>40
<211>20
<212>DNA
<213>人工序列
<400>40
gagacagccctgtgctgaag20
<210>41
<211>25
<212>DNA
<213>人工序列
<400>41
catcacactatcctgacatgaacaa25
<210>42
<211>25
<212>DNA
<213>人工序列
<400>42
agaattaaatgaaccactgcacctg25
<210>43
<211>25
<212>DNA
<213>人工序列
<400>43
acaggtacattcacttaacaggctc25
<210>44
<211>21
<212>DNA
<213>人工序列
<400>44
gttactaagcccatgccatcg21
<210>45
<211>22
<212>DNA
<213>人工序列
<400>45
gatgacttcccagagctccaga22
<210>46
<211>22
<212>DNA
<213>人工序列
<400>46
gatacaagagcttccggagacc22
<210>47
<211>22
<212>DNA
<213>人工序列
<400>47
ccttctgtctcaccttctttcg22
<210>48
<211>21
<212>DNA
<213>人工序列
<400>48
caaagggaccaagtcaagagc21
<210>49
<211>23
<212>DNA
<213>人工序列
<400>49
gttcccacttgttctttttctgc23
<210>50
<211>21
<212>DNA
<213>人工序列
<400>50
gactgcactttgcgaaacatg21
<210>51
<211>25
<212>DNA
<213>人工序列
<400>51
tgagaggagaagactgtgtgtttta25
<210>52
<211>22
<212>DNA
<213>人工序列
<400>52
accaggcatttgaccttctagc22
<210>53
<211>23
<212>DNA
<213>人工序列
<400>53
cccaaggtaggaccagaagtgtt23
<210>54
<211>22
<212>DNA
<213>人工序列
<400>54
ttggataccatccccaagacat22
<210>55
<211>25
<212>DNA
<213>人工序列
<400>55
aatggtgagaggttgatggtaaaat25
<210>56
<211>23
<212>DNA
<213>人工序列
<400>56
agtgagaagtgtcttgggttgga23
<210>57
<211>21
<212>DNA
<213>人工序列
<400>57
tgtccaccaagggaacgtgag21
<210>58
<211>23
<212>DNA
<213>人工序列
<400>58
ggctgatgtgtttggtgagctgt23
<210>59
<211>23
<212>DNA
<213>人工序列
<400>59
ggttgtttctctagccagacagg23
<210>60
<211>22
<212>DNA
<213>人工序列
<400>60
accatgacccccagcaatttgt22
<210>61
<211>25
<212>DNA
<213>人工序列
<400>61
ccaggatgaaagttcacttcagatg25
<210>62
<211>26
<212>DNA
<213>人工序列
<400>62
ccatagatctcatagccatcctttta26
<210>63
<211>22
<212>DNA
<213>人工序列
<400>63
agttgctcacacattcacctcc22
<210>64
<211>22
<212>DNA
<213>人工序列
<400>64
cccaacacaaatgcttcccaac22
<210>65
<211>28
<212>DNA
<213>人工序列
<400>65
ttaggtcacttataaacttttggttctg28
<210>66
<211>27
<212>DNA
<213>人工序列
<400>66
ggacaaatagaaaaaagaaagccacac27
<210>67
<211>23
<212>DNA
<213>人工序列
<400>67
tacattcatttctgcatgggtgg23
<210>68
<211>25
<212>DNA
<213>人工序列
<400>68
cagaactgtgatcacctaatagcca25
<210>69
<211>24
<212>DNA
<213>人工序列
<400>69
ccacccatgagaaatatatccaca24
<210>70
<211>27
<212>DNA
<213>人工序列
<400>70
attcattcaccatttgatagccatttg27
<210>71
<211>25
<212>DNA
<213>人工序列
<400>71
gtggaagagaaatgcccaaaagact25
<210>72
<211>21
<212>DNA
<213>人工序列
<400>72
catgccacaacctgcttcact21
<210>73
<211>26
<212>DNA
<213>人工序列
<400>73
aaccaaagatcctaagtctgatcaat26
<210>74
<211>25
<212>DNA
<213>人工序列
<400>74
ctgatgatgtgcaagaaaggtaggt25
<210>75
<211>22
<212>DNA
<213>人工序列
<400>75
tttcaaatagcaatggctcgtc22
<210>76
<211>25
<212>DNA
<213>人工序列
<400>76
gggatgtcccgtcttattaatgaac25
<210>77
<211>25
<212>DNA
<213>人工序列
<400>77
taagtgggacagttaagagaaggct25
<210>78
<211>25
<212>DNA
<213>人工序列
<400>78
cactcaacacacagaaacatcaagc25
<210>79
<211>27
<212>DNA
<213>人工序列
<400>79
acatcatcattcataattgtgattgag27
<210>80
<211>26
<212>DNA
<213>人工序列
<400>80
gaacactaaacaagacgaaaaaccct26
<210>81
<211>26
<212>DNA
<213>人工序列
<400>81
aatttaggtgatttttcccatgatga26
<210>82
<211>26
<212>DNA
<213>人工序列
<400>82
agcaaagtattgtgataacagtctcc26
<210>83
<211>20
<212>DNA
<213>人工序列
<400>83
caaacattcccgcctcacat20
<210>84
<211>20
<212>DNA
<213>人工序列
<400>84
ggcaactggggctctgatct20
<210>85
<211>24
<212>DNA
<213>人工序列
<400>85
tttactttgtaccaggggtgtttc24
<210>86
<211>24
<212>DNA
<213>人工序列
<400>86
ctcctttgttaatttctgtctgcc24
<210>87
<211>20
<212>DNA
<213>人工序列
<400>87
aacctcttttgtgcctcccc20
<210>88
<211>24
<212>DNA
<213>人工序列
<400>88
ttgttgatgatagtggcaaaggag24
<210>89
<211>22
<212>DNA
<213>人工序列
<400>89
ctggacaccagaccaaaaacaa22
<210>90
<211>23
<212>DNA
<213>人工序列
<400>90
caagtttgttggcttcttttgag23
<210>91
<211>26
<212>DNA
<213>人工序列
<400>91
cactctgtacaaatcagatgaagcct26
<210>92
<211>20
<212>DNA
<213>人工序列
<400>92
gaagccaagcgactcctacg20
<210>93
<211>29
<212>DNA
<213>人工序列
<400>93
cttgggtcatctctatcatagaacttatc29
<210>94
<211>22
<212>DNA
<213>人工序列
<400>94
gaacaacttactggcagggcac22
<210>95
<211>21
<212>DNA
<213>人工序列
<400>95
aggaagctctcccgagttctc21
<210>96
<211>21
<212>DNA
<213>人工序列
<400>96
gtccgattgtccctctctggt21
<210>97
<211>25
<212>DNA
<213>人工序列
<400>97
ttgggttaattttgctcagagtatc25
<210>98
<211>22
<212>DNA
<213>人工序列
<400>98
agccacagagtgccctagtcag22
<210>99
<211>22
<212>DNA
<213>人工序列
<400>99
gtctggaagttcgtcaaattgc22
<210>100
<211>21
<212>DNA
<213>人工序列
<400>100
cgtacgacatcatcacagcca21
<210>101
<211>26
<212>DNA
<213>人工序列
<400>101
ctcaagaaggcttggttacaagatcc26
<210>102
<211>29
<212>DNA
<213>人工序列
<400>102
tgtgtttgaatggtttcctacttattgac29
<210>103
<211>24
<212>DNA
<213>人工序列
<400>103
tgtcctctgagatgatgaatgctt24
<210>104
<211>21
<212>DNA
<213>人工序列
<400>104
gagtttgggggtccatgctag21
<210>105
<211>30
<212>DNA
<213>人工序列
<400>105
gatagaagaatgaatttgaaaaagcatcag30
<210>106
<211>28
<212>DNA
<213>人工序列
<400>106
ataatatcctttctgttttgtccatctg28
<210>107
<211>22
<212>DNA
<213>人工序列
<400>107
gtgaagagaggcaaggcaaaac22
<210>108
<211>20
<212>DNA
<213>人工序列
<400>108
acagaacaggacccaggcag20
<210>109
<211>24
<212>DNA
<213>人工序列
<400>109
gtgatggatgtaaacacagtgcct24
<210>110
<211>24
<212>DNA
<213>人工序列
<400>110
tcctgtcaacattgatagcctgag24
<210>111
<211>20
<212>DNA
<213>人工序列
<400>111
cctcccacaccagtttctcc20
<210>112
<211>20
<212>DNA
<213>人工序列
<400>112
gaaaaacactaacctgtcct20
<210>113
<211>24
<212>DNA
<213>人工序列
<400>113
tctggccttacatatgctacactt24
<210>114
<211>22
<212>DNA
<213>人工序列
<400>114
gctcattctttgttgtcccctc22
<210>115
<211>25
<212>DNA
<213>人工序列
<400>115
agagcagcacactgaggctttatgg25
<210>116
<211>29
<212>DNA
<213>人工序列
<400>116
tttttgtcccacctccccaccctgttcct29
<210>117
<211>31
<212>DNA
<213>人工序列
<400>117
tttttttttccagatagagctaaaactgaag31
<210>118
<211>33
<212>DNA
<213>人工序列
<400>118
ttttttttttagttgcgtttacaactttctccc33
<210>119
<211>35
<212>DNA
<213>人工序列
<400>119
ttttttttttgactaaattgttgaacactggttac35
<210>120
<211>38
<212>DNA
<213>人工序列
<400>120
ttttttttttttttttttaaacccagcacctaccctca38
<210>121
<211>38
<212>DNA
<213>人工序列
<400>121
tttttttttttttttttttccttttgtgattccacttc38
<210>122
<211>40
<212>DNA
<213>人工序列
<400>122
tctctctctctctcttctcatcctgttattatttgtttac40
<210>123
<211>42
<212>DNA
<213>人工序列
<400>123
tttttttttttttttttcccaaggtcctgtgacctgagtaaa42
<210>124
<211>46
<212>DNA
<213>人工序列
<400>124
tttttttttttttttttttttttgcaaacaaagactgaaaaggtga46
<210>125
<211>49
<212>DNA
<213>人工序列
<400>125
tttttttttttttttttttttttttttttattcacctctagtccctctg49
<210>126
<211>49
<212>DNA
<213>人工序列
<400>126
tttttttttttttttttttttttttttggaaagacgaaaggaatcttga49
<210>127
<211>52
<212>DNA
<213>人工序列
<400>127
tttttttttttttttttttttttttttttcattctccttcttctatctgtat52
<210>128
<211>53
<212>DNA
<213>人工序列
<400>128
ctctctctctctctctctctctctctcagtgtcaattttgaccagatattaaa53
<210>129
<211>56
<212>DNA
<213>人工序列
<400>129
tttttttttttttttttttttttttttttttttttagaagagcctcaaggacagcc56
<210>130
<211>56
<212>DNA
<213>人工序列
<400>130
ttttttttttttttttttttttttttttttttttttttatctgactccccacagcc56
<210>131
<211>60
<212>DNA
<213>人工序列
<400>131
ttttttttttttttttttttttttttttttttttttttttaacaggctctctttccaccc60
<210>132
<211>60
<212>DNA
<213>人工序列
<400>132
ttttttttttttttttttttttttttttttttttttttaatgtaaaaactgcaagtggtt60
<210>133
<211>64
<212>DNA
<213>人工序列
<400>133
tttttttttttttttttttttttttttttttttttttttttttccagaagcaactccagc60
acac64
<210>134
<211>65
<212>DNA
<213>人工序列
<400>134
tttttttttttttttttttttttttttttttttttttttctcaccttctttcgtgtgcct60
gtgca65
<210>135
<211>67
<212>DNA
<213>人工序列
<400>135
tttttttttttttttttttttttttttttttttttttttttttttcagcatttaaaaata60
aaaccga67
<210>136
<211>70
<212>DNA
<213>人工序列
<400>136
tttttttttttttttttttttttttttttttttttttttttttgaaagaacatcttaata60
ctatcataac70
<210>137
<211>73
<212>DNA
<213>人工序列
<400>137
tttttttttttttttttttttttttttttttttttttttttttttttttttggataccat60
ccccaagacatct73
<210>138
<211>78
<212>DNA
<213>人工序列
<400>138
tttttttttttttttttttttttttttttttttttttttttttttttttttttttaaggg60
aacgtgaggaggccacac78
<210>139
<211>76
<212>DNA
<213>人工序列
<400>139
ctctctctctctctctctctctctctctctctctctctctctctctctctctggaactga60
ggattttcaatttcct76
<210>140
<211>19
<212>DNA
<213>人工序列
<400>140
tctagccagacagggcagg19
<210>141
<211>26
<212>DNA
<213>人工序列
<400>141
cttctcatagccatccttttattctc26
<210>142
<211>24
<212>DNA
<213>人工序列
<400>142
tttcacctcctttgggtcttcatt24
<210>143
<211>29
<212>DNA
<213>人工序列
<400>143
aattaatcaggccattgcctttaagattg29
<210>144
<211>39
<212>DNA
<213>人工序列
<400>144
ctctctctctctctctatcacctaatagccagcgatagc39
<210>145
<211>36
<212>DNA
<213>人工序列
<400>145
tctctctctcacccatgagaaatatatccacaaaaa36
<210>146
<211>35
<212>DNA
<213>人工序列
<400>146
ctctctctcttcactatatgttggccttgattggt35
<210>147
<211>38
<212>DNA
<213>人工序列
<400>147
ctctctctctctgatgtgcaagaaaggtaggtaaaaac38
<210>148
<211>40
<212>DNA
<213>人工序列
<400>148
tttttttttttttttttttggctcgtctatggttagtctc40
<210>149
<211>44
<212>DNA
<213>人工序列
<400>149
ctctctctctctctctctctctgaaacatcaagctgagctctgc44
<210>150
<211>44
<212>DNA
<213>人工序列
<400>150
tctctctctctctctctcatcatcattcataattgtgattgagt44
<210>151
<211>45
<212>DNA
<213>人工序列
<400>151
ctctctctctctctctctcagtttgcactaaatgattacaggtta45
<210>152
<211>48
<212>DNA
<213>人工序列
<400>152
tctctctctctctctctctctctctctctggctctgatctgacggcaa48
<210>153
<211>50
<212>DNA
<213>人工序列
<400>153
tttttttttttttttttttttttttttttctgccacataaggatgaaact50
<210>154
<211>53
<212>DNA
<213>人工序列
<400>154
ctctctctctctctctctctctctctctctgtgtaccaccttctctgtcacca53
<210>155
<211>52
<212>DNA
<213>人工序列
<400>155
tctctctctctctctctctctctctctgtatctgttcatgtattttgctgat52
<210>156
<211>55
<212>DNA
<213>人工序列
<400>156
ttttttttttttttttttttttttttttttattcgtatagtcagtgtggtcagag55
<210>157
<211>58
<212>DNA
<213>人工序列
<400>157
tttttttttttttttttttttttttttttttttttaaggatgtggaagtctagtgtga58
<210>158
<211>63
<212>DNA
<213>人工序列
<400>158
tctctctctctctctctctctctctctctctctctctctctaggaagctctcccgagttc60
tct63
<210>159
<211>65
<212>DNA
<213>人工序列
<400>159
tctctctctctctctctctctctctctctctctctctctgtgggttaattttgctcagag60
tatcc65
<210>160
<211>66
<212>DNA
<213>人工序列
<400>160
ctctctctctctctctctctctctctctctctctctctctctctctgctatgtggcatac60
ttggac66
<210>161
<211>68
<212>DNA
<213>人工序列
<400>161
tctctctctctctctctctctctctctctctctctctcttgtgtttgaatggtttcctac60
ttattgac68
<210>162
<211>71
<212>DNA
<213>人工序列
<400>162
tctctctctctctctctctctctctctctctctctctctctctctctctctctttctgag60
ccctcagcaaa71
<210>163
<211>74
<212>DNA
<213>人工序列
<400>163
tctctctctctctctctctctctctctctctctctctctctctgaaattctaatttttct60
tatctttgttttaa74
<210>164
<211>77
<212>DNA
<213>人工序列
<400>164
ctctctctctctctctctctctctctctctctctctctctctctctctctctctctctgc60
caacctctccagattgc77
<210>165
<211>73
<212>DNA
<213>人工序列
<400>165
ctctctctctctctctctctctctctctctctctctctctctctctctctgcctgagaat60
gtgagttctacaa73
Claims (4)
1.55个SNP位点多色荧光复合检测的试剂盒,其包括a)扩增体系:PCR缓冲溶液、MgCl2、dNTPs、PCR引物、TaqDNA聚合酶、模板DNA,b)扩增产物纯化试剂:核酸外切酶Ⅰ、虾碱性磷酸酶,c)延伸反应试剂:延伸引物、SNaPshot反应混合液,d)延伸产物纯化试剂:虾碱性磷酸酶,e)测试试剂:Hi-Di甲酰胺和GeneScanTMSizeStandards-120LIZ,其特征在于,所述PCR引物和延伸引物由55个SNP基因位点的引物组成,所述55个SNP基因位点在NCBI中SNP数据库的rs号分别为rs1109037,rs3780962,rs6591147,rs2342747,rs9951171,rs13182883,rs10092491,rs6955448,rs6444724,rs430046,rs321198,rs159606,rs560681,rs214955,rs13218440,rs9905977,rs1821380,rs279844,rs8078417,rs4606077,rs445251,rs987640,rs4530059,rs338882,rs10488710,rs6811238,rs722290,rs7041158,rs7520386,rs9866013,rs1498553,rs2270529,rs1478829,rs1523537,rs4364205,rs985492,rs12997453,rs740598,rs1736442,rs1294331,rs993934,rs10776839,rs315791,rs576261,rs2399332,rs2272998,rs7205345,rs2269355,rs1058083,rs10773760,rs464663,rs221956,rs1336071,rs1027895和位于人类牙釉质蛋白基因Amelogeningene上的一个性别检测SNP位点,所述性别检测SNP位点在女性个体中为纯合子T/T,在男性个体中为杂合子C/T,性别检测SNP位点的5’侧翼序列为TTTTACCTTCTTCTTTCT,3’侧翼序列为TTGTAGAACTCACATTCTCAGGC。
2.根据权利要求1所述的55个SNP位点多色荧光复合检测的试剂盒,其特征在于所述55个SNP基因位点的PCR引物和延伸引物的序列为:
rs1109037,PCR引物SEQIDNO.1-2,延伸引物SEQIDNO.111;
rs3780962,PCR引物SEQIDNO.3-4,延伸引物SEQIDNO.112;
rs6591147,PCR引物SEQIDNO.5-6,延伸引物SEQIDNO.113;
rs2342747,PCR引物SEQIDNO.7-8,延伸引物SEQIDNO.114;
rs9951171,PCR引物SEQIDNO.9-10,延伸引物SEQIDNO.115;
rs13182883,PCR引物SEQIDNO.11-12,延伸引物SEQIDNO.116;
rs10092491,PCR引物SEQIDNO.13-14,延伸引物SEQIDNO.117;
rs6955448,PCR引物SEQIDNO.15-16,延伸引物SEQIDNO.118;
rs6444724,PCR引物SEQIDNO.17-18,延伸引物SEQIDNO.119;
rs430046,PCR引物SEQIDNO.19-20,延伸引物SEQIDNO.120;
rs321198,PCR引物SEQIDNO.21-22,延伸引物SEQIDNO.121;
rs159606,PCR引物SEQIDNO.23-24,延伸引物SEQIDNO.122;
rs560681,PCR引物SEQIDNO.25-26,延伸引物SEQIDNO.123;
rs214955,PCR引物SEQIDNO.27-28,延伸引物SEQIDNO.124;
rs13218440,PCR引物SEQIDNO.29-30,延伸引物SEQIDNO.125;
rs9905977,PCR引物SEQIDNO.31-32,延伸引物SEQIDNO.126;
rs1821380,PCR引物SEQIDNO.33-34,延伸引物SEQIDNO.127;
rs279844,PCR引物SEQIDNO.35-36,延伸引物SEQIDNO.128;
rs8078417,PCR引物SEQIDNO.37-38,延伸引物SEQIDNO.129;
rs4606077,PCR引物SEQIDNO.39-40,延伸引物SEQIDNO.130;
rs445251,PCR引物SEQIDNO.41-42,延伸引物SEQIDNO.131;
rs987640,PCR引物SEQIDNO.43-44,延伸引物SEQIDNO.132;
rs4530059,PCR引物SEQIDNO.45-46,延伸引物SEQIDNO.133;
rs338882,PCR引物SEQIDNO.47-48,延伸引物SEQIDNO.134;
rs10488710,PCR引物SEQIDNO.49-50,延伸引物SEQIDNO.135;
rs6811238,PCR引物SEQIDNO.51-52,延伸引物SEQIDNO.136;
rs722290,PCR引物SEQIDNO.53-54,延伸引物SEQIDNO.137;
rs7041158,PCR引物SEQIDNO.55-56,延伸引物SEQIDNO.138;
rs7520386,PCR引物SEQIDNO.57-58,延伸引物SEQIDNO.139;
rs9866013,PCR引物SEQIDNO.59-60,延伸引物SEQIDNO.140;
rs1498553,PCR引物SEQIDNO.61-62,延伸引物SEQIDNO.141;
rs2270529,PCR引物SEQIDNO.63-64,延伸引物SEQIDNO.142;
rs1478829,PCR引物SEQIDNO.65-66,延伸引物SEQIDNO.143;
rs1523537,PCR引物SEQIDNO.67-68,延伸引物SEQIDNO.144;
rs4364205,PCR引物SEQIDNO.69-70,延伸引物SEQIDNO.145;
rs985492,PCR引物SEQIDNO.71-72,延伸引物SEQIDNO.146;
rs12997453,PCR引物SEQIDNO.73-74,延伸引物SEQIDNO.147;
rs740598,PCR引物SEQIDNO.75-76,延伸引物SEQIDNO.148;
rs1736442,PCR引物SEQIDNO.77-78,延伸引物SEQIDNO.149;
rs1294331,PCR引物SEQIDNO.79-80,延伸引物SEQIDNO.150;
rs993934,PCR引物SEQIDNO.81-82,延伸引物SEQIDNO.151;
rs10776839,PCR引物SEQIDNO.83-84,延伸引物SEQIDNO.152;
rs315791,PCR引物SEQIDNO.85-86,延伸引物SEQIDNO.153;
rs576261,PCR引物SEQIDNO.87-88,延伸引物SEQIDNO.154;
rs2399332,PCR引物SEQIDNO.89-90,延伸引物SEQIDNO.155;
rs2272998,PCR引物SEQIDNO.91-92,延伸引物SEQIDNO.156;
rs7205345,PCR引物SEQIDNO.93-94,延伸引物SEQIDNO.157;
rs2269355,PCR引物SEQIDNO.95-96,延伸引物SEQIDNO.158;
rs1058083,PCR引物SEQIDNO.97-98,延伸引物SEQIDNO.159;
rs10773760,PCR引物SEQIDNO.99-100,延伸引物SEQIDNO.160;
rs464663,PCR引物SEQIDNO.101-102,延伸引物SEQIDNO.161;
rs221956,PCR引物SEQIDNO.103-104,延伸引物SEQIDNO.162;
rs1336071,PCR引物SEQIDNO.105-106,延伸引物SEQIDNO.163;
rs1027895,PCR引物SEQIDNO.107-108,延伸引物SEQIDNO.164;
位于人类牙釉质蛋白基因Amelogeningene上的一个性别检测SNP位点,PCR引物SEQIDNO.109-110,延伸引物SEQIDNO.165。
3.根据权利要求1所述的55个SNP位点多色荧光复合检测的试剂盒,其特征在于同时复合扩增包含55个SNPs位点的DNA片段。
4.利用如权利要求1所述的55个SNP位点多色荧光复合检测的试剂盒进行个体识别的测试方法,其特征在于按照下列步骤进行:
a)提取DNA,得DNA提取液;
b)在扩增体系中加入步骤a)所得的DNA提取液,然后在扩增仪中进行扩增,扩增条件为:95℃、5min预变性;然后依次在95℃、30s,60℃、30s,65℃、30s,进行35个循环;再在65℃保持7min;最终在4℃保存,获得扩增产物;
c)应用核酸外切酶Ⅰ和虾碱性磷酸酶纯化扩增产物,纯化反应条件:37℃、1h,然后80℃、15min,最终在4℃保存,得纯化后的扩增产物;
d)对纯化后的扩增产物进行延伸反应
将纯化后的扩增产物加入至所述的延伸反应试剂中进行延伸反应;所述的延伸反应的条件:依次在96℃、10s,58℃、5s,60℃、30s进行50个循环,最终保存在4℃,得延伸产物;
e)应用虾碱性磷酸酶纯化延伸产物
将虾碱性磷酸酶加入延伸产物中,在下列条件下进行纯化反应:37℃、1h,80℃、15min,最终保存在4℃,得纯化后的延伸产物;
f)检测、确定产物,
将步骤e)中得到的纯化后的延伸产物中加入至所述的测试试剂中,混匀;然后95℃变性3min,4℃迅速冷却后进行电泳检测;最后根据延伸产物峰的位置和颜色确定55个SNP基因位点的基因型。
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CN108342489A (zh) * | 2017-01-23 | 2018-07-31 | 公安部物证鉴定中心 | 一种对男性个体进行y-snp分型的方法和*** |
CN113564266A (zh) * | 2021-09-24 | 2021-10-29 | 上海仁东医学检验所有限公司 | Snp分型遗传标记组合、检测试剂盒及用途 |
CN113564266B (zh) * | 2021-09-24 | 2021-12-17 | 上海仁东医学检验所有限公司 | Snp分型遗传标记组合、检测试剂盒及用途 |
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