CN113684269B - 一种家族性高胆固醇血症基因检测文库、其构建方法和试剂盒 - Google Patents
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Abstract
本发明属于基因检测技术领域，公开了一种家族性高胆固醇血症基因检测文库、及其构建方法和试剂盒。构建方法包括步骤：1)获得DNA样本；2)利用引物组的引物对所述DNA样本进行第1轮多重PCR扩增，得到扩增产物；3)合并第1轮多重PCR反应得到的扩增产物，在扩增产物中加入接头引物1和接头引物2，然后进行第2轮接头序列PCR反应，得到基因检测文库。本发明构建方法基因panel扩大，检测范围更加全面，测序覆盖度达到100％；构建的基因检测文库适用于DNBSEQ测序平台。
Description
技术领域
本发明涉及基因检测技术领域，具体涉及一种家族性高胆固醇血症基因检测文库、其构建方法和试剂盒。
背景技术
家族性高胆固醇血症(familial hypercholesterolemia,FH)是一种常染色体显性遗传病，病理特征为血浆总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)水平增高及早发冠心病(PCAD)。冠状动脉粥样硬化性心脏病(CAD)的病理基础是动脉粥样硬化(AS)，而FH因血脂代谢异常会间接或直接促发AS，与PCAD(CAD发病年龄男性＜55岁，女性＜65岁)的发生、发展具有密切关系。FH患者血脂代谢失衡会加快AS及PCAD进程，早期发现和诊断FH，及早进行干预，可显著降低冠脉疾病的发病率及死亡率，改善临床预后。
目前已知的FH相关变异超过2000个，其中约1000个变异已经得到足够证据支持而被归类为致病变异和可能致病变异两类之中，这些变异主要分布在三个基因：LDLR(90％以上)、APOB(5％～10％)和PCSK9(1％以下)。但是，随着基因测序技术的发展，以及医患对于FH认知的不断提高，越来越多的基因，以及基因的突变位点被发现，对FH的相关基因检测的覆盖度还有待于进一步改善，例如授权公告号为CN109517884B的中国发明专利公开了FH的测序文库，仅涉及LDLR、APOB、PCSK9这三种基因，覆盖度有待于进一步提高，并且采用的是Ion Torrent测序平台。
与目前非常成熟，并且已经面临要被更新迭代的Ion Torrent测序平台和Illumina测序平台相比，DNBSEQ测序平台主要定位于医院、检验所级别的中小型规模的测序应用场景，仪器成本低，各项性能指标更加优异，但是目前还未见基于DNBSEQ测序平台的FH基因检测文库的相关报道。
发明内容
本发明提供一种家族性高胆固醇血症基因检测文库、其构建方法和试剂盒，以至少解决上述技术问题之一。
本发明实现其目的采用的技术方案是：
一种家族性高胆固醇血症基因检测文库试剂盒，包含引物组3，引物组3的序列为SEQ ID NO.323-382，检测如下基因或基因位点：LDLRAP1基因，CELSR2基因的多态性位点rs629301，ABCG8基因的多态性位点rs4299376，SLC22A1基因的多态性位点rs1564348，HFE基因的多态性位点rs1800562，MYLIP基因的多态性位点rs3757354，ST3GAL4基因的多态性位点rs11220462，NYNRIN基因的多态性位点rs8017377，APOE基因的多态性位点rs429358、rs7412，LDLR基因的多态性位点rs6511720，APOB基因的多态性位点rs1367117，PCSK9基因的多态性位点rs2479409。
本发明还提供另一种家族性高胆固醇血症基因检测文库试剂盒，包含引物组1，引物组1的序列为SEQ ID NO.1-152，检测如下基因：LDLRAP1基因、LDLR基因，APOB基因，PCSK9基因。
本发明还提供第三种家族性高胆固醇血症基因检测文库试剂盒，包含引物组2，引物组2的序列为SEQ ID NO.153-322，检测如下基因：LDLR基因，APOB基因，PCSK9基因，LDLRAP1基因。
上述的任意一种家族性高胆固醇血症基因检测文库试剂盒，还包括测序接头引物1和接头引物2，所述接头引物1的序列为SEQ ID NO.383所示，所述接头引物2的序列为SEQID NO.384所示。
本发明提供的一种家族性高胆固醇血症基因检测文库的构建方法，包括如下步骤：
1)获得DNA样本；
2)采用权利要求1所述的试剂盒中的引物组3，权利要求2所述的试剂盒中的引物组1，权利要求3所述的试剂盒中的引物组2中的任一组，任意两组，或三组对所述DNA样本进行第1轮多重PCR反应得到扩增产物；
3)合并第1轮多重PCR反应得到的扩增产物，在扩增产物中加入接头引物1和接头引物2，然后进行第2轮接头序列PCR反应，得到基因检测文库。
在上述技术方案中，所述步骤3)中，当引物组1-3分别进行第1轮多重PCR反应时，引物组1-3的多重PCR扩增产物以体积比6-8﹕5-7﹕1-3合并。
在上述技术方案中，引物组1和引物组2的第1轮多重PCR反应的反应条件如下：
引物组3的第1轮多重PCR反应的反应条件如下：
本发明提供的一种家族性高胆固醇血症基因检测文库，所述基因检测文库由上述任一项所述的构建方法制备而成。
本发明的有益效果在于：
(1)本发明设计了适用DNBSEQ测序平台的关于FH基因检测的191对引物对(SEQ IDNO.1-382)，其中引物SEQ ID NO.1-324，以及引物SEQ ID NO.347-382用于检测LDLRAP1基因、PCSK9基因、LDLR基因和APOB基因的编码外显子及侧翼区(±10bp)，主要考虑的是单基因遗传的突变特点，测序覆盖度已经能够达到100％。
(2)除了考虑单基因遗传的突变特点外，本发明还考虑到了多基因遗传病的突变特点，将CELSR2基因的多态性位点(rs629301)，ABCG8基因的多态性位点(rs4299376)，SLC22A1基因的多态性位点(rs1564348)，HFE基因的多态性位点(rs1800562)，MYLIP基因的多态性位点(rs3757354)，ST3GAL4基因的多态性位点(rs11220462)，NYNRIN基因的多态性位点(rs8017377)，APOE基因的多态性位点(rs429358、rs7412)，LDLR基因的多态性位点(rs6511720)，APOB基因的多态性位点(rs1367117)，PCSK9基因的多态性位点(rs2479409)纳入到检测范围，进一步提高了基因检测文库的覆盖范围。
本发明提供的多重靶向捕获试剂盒能够检测FH相关基因变异的多个位点，基因panel扩大，检测范围更加全面，测序覆盖度能够达到100％，不需要使用探针捕获，利用多重PCR扩增实现待测区域的高度覆盖，具有高检测覆盖度和高均一性，样本消耗量少、样本要求低。
(3)三组引物组，主要分组依据在于GC含量，以及防止引物之间相互配对，其中，引物组1和引物组2的GC含量大致范围在40-60％，引物组3的GC含量大致范围在70-80％。
(4)本发明提供的试剂盒可以采用多种样本进行文库构建，如唾液gDNA、血液gDNA等。本发明优化了流程步骤，将多重PCR完成后的引物消化步骤改为磁珠纯化，减少了实验用时。
(5)除引物外，检测时，本发明所采用的第1轮多重PCR反应和第2轮接头序列PCR反应的检测条件，使构建的基因检测文库具有构建周期短，比对率高，捕获率高，均一性好，重复性好，操作简便等优点。
(6)本发明所称的“覆盖度”是指达到指定深度的测序碱基占有整个基因组或目标区域的百分比；深度是指比对到已知参考序列的碱基平均测序次数。
附图说明
图1是采用Agilent 2200生物分析仪对基因检测文库进行文库片段长度质控和浓度质控结果图。
具体实施方式
下面结合实施例对本发明作进一步说明，但并不因此而限制本发明。下述实施例中的实验方法，如无特别说明，均为常规方法。下述实施例中所使用的材料、试剂等，如无特殊说明，均可通过商业途径获得。
试剂来源：
以下实施例所涉及的自备试剂和仪器如下表1所示：
表1自备试剂和仪器
实施例1、采用家族性高胆固醇血症多重靶向捕获试剂盒构建基因检测文库
一、家族性高胆固醇血症多重靶向捕获试剂盒组成
表2试剂盒组成
二、家族性高胆固醇血症多重靶向捕获试剂盒检测方法
1.获取DNA样本
对收集的人类全血EDTA抗凝样本进行全基因组的DNA提取，对DNA的浓度和纯度进行检测，要求浓度＞5.6ng/μL，纯度OD260/280为1.8-2.0。
2.构建基因检测文库
1)第1轮多重PCR反应
本实施例所涉及的FH相关基因有4个基因和12个多态位点，4个基因、12个多态位点以及对应的PCR扩增引物组如下：
根据LDLR(低密度脂蛋白受体,Low-density lipoprotein receptor)基因所设计的引物：序列SEQ ID NO.33-58和序列SEQ ID NO.201-220；
根据APOB(载脂蛋白B,apolipoprotein B)基因所设计的引物：SEQ ID NO.序列59-152，序列SEQ ID NO.221-322和序列SEQ ID NO.347-366；
根据PCSK9(人蛋白原转化酶枯草杆菌蛋白酶9,proprotein convertasesubtilisin/kexintype9)基因所设计的引物：序列SEQ ID NO.15-32、序列SEQ ID NO.167-200和序列SEQ ID NO.367-382；
根据LDLRAP1(低密度脂蛋白受体衔接蛋白1基因,(low density lipoproteinreceptor adaptor protein 1)基因所设计的引物：序列SEQ ID NO.1-14、序列SEQ IDNO.153-166和序列SEQ ID NO.323-324；
根据PCSK9基因的多态性位点(rs2479409)所设计的引物：序列SEQ ID NO.325-326；
根据CELSR2(钙粘蛋白EGF-LAG七通型受体2基因，cadherin EGF-LAG seven-passG-type receptor 2)基因的多态性位点(rs629301)所设计的引物：序列SEQ ID NO.327-328；
根据ST3GAL4(唾液酸转移酶4)基因的多态性位点(rs11220462)所设计的引物：序列SEQ ID NO.329-330；
根据NYNRIN(NYN domain and retroviral integrase containing)基因的多态性位点(rs8017377)所设计的引物：序列SEQ ID NO.331-332；
根据LDLR基因的多态性位点(rs6511720)所设计的引物：序列SEQ ID NO.333-334；
根据APOE(apolipoprotein E，ApoE)基因的多态性位点(rs429358、rs7412)所设计的引物：序列SEQ ID NO.335-336；
根据APOB基因的多态性位点(rs1367117)所设计的引物：序列SEQ ID NO.337-338；
根据ABCG8(ATP结合盒B亚族成员8转运蛋白基因)基因的多态性位点(rs4299376)所设计的引物：序列SEQ ID NO.339-340；
根据MYLIP(肌球蛋白调节轻链相互作用蛋白,myosin regulatory light chaininteracting protein)基因的多态性位点(rs3757354)所设计的引物：序列SEQ IDNO.341-342；
根据HFE(血色沉着病基因,hemochromatosis)基因的多态性位点(rs1800562)所设计的引物：序列SEQ ID NO.343-344；
根据SLC22A1(阳离子转运蛋白1)基因的多态性位点(rs1564348)所设计的引物：序列SEQ ID NO.345-346。
上述引物分为引物组1、引物组2、引物组3。引物组1所含引物的核苷酸序列如下表3所列：
表3引物组1所含引物的核苷酸序列
引物组2所含引物的核苷酸序列如下表4所列：
表4引物组2所含引物的核苷酸序列
引物组3所含引物的核苷酸序列如下表5所列：
表5引物组3所含引物的核苷酸序列
多重PCR反应分为3个反应管，引物组1反应管、引物组2反应管、引物组3反应管中除了引物不一样，其它试剂都一样，多重PCR反应体系(总体系30μL)如下表6所示。
表6.第1轮多重PCR反应体系
| 组份 | 加入量(μL) |
| ddH<sub>2</sub>O | 9-x |
| Enhancer buffer NB(1N) | 3.5 |
| Enhancer buffer M | 2.5 |
| 引物组1/引物组2/引物组3 | 5 |
| gDNA | x |
| IGT-EM808polymerase mixture | 10 |
gDNA起始量均为50ng/反应管；DNA的浓度为Qubit dsDNA HS Assay Kit(ThermoFisher)的定量结果；引物组中的各引物的浓度为：10μM。
将反应混合物用漩涡振荡器震荡混匀，迷你离心机离心5秒置于PCR仪中反应。反应条件如表7(引物组1和2)和表8(引物组3)中所示。
注意事项：①需要严格按照表7和表8中的反应体系和反应条件进行PCR反应，更改反应参数可能会导致文库的质量下降或文库构建失败；②进行PCR反应前，先将所有gDNA的浓度稀释到同一浓度，并转移到PCR 8联管中，一方面是便于排枪操作，另一方面是为了加入相同起始量的gDNA，降低最终文库的浓度差异，便于混合文库；③执行多重PCR反应时，特别是样本量多的情况下，请将引物组1设为一组，引物组2设为一组，引物组3设为一组，便于制备反应试剂混合液和排枪操作；④执行多重PCR反应时，请将解冻好的试剂放置在冰盒上，并在冰盒上进行加样操作；⑤请在PCR管壁上方或者管盖上进行反应编号标记，防止高温或其他原因导致记号消失，避免后续产物混合操作失误，造成样本交叉污染。
请按照普通PCR的操作规范进行第1轮多重PCR反应
表7.第1轮多重PCR反应条件(引物组1和引物组2对应的反应管)
表8.第1轮多重PCR反应条件(引物组3对应的反应管)
2)第1轮多重PCR产物合并
多重PCR反应结束后，3个反应管的PCR产物先分别加入接头引物1(SEQ IDNO.383)和接头引物2(SEQ ID NO.384)进行连接反应，反应完毕后3个反应管的PCR产物按照表9进行合并，总体积为30μL。
表9.多重PCR产物合并
| 反应管编号 | T1 | T2 | T3 |
| 混合体积 | 14μL | 12μL | 4μL |
向30μL PCR合并产物加入27μL室温平衡后的AMPure XP磁珠，用移液器轻缓吸打混匀20次；
室温孵育5min后，将PCR管置于DynaMag-96Side磁力架上3min；
彻底移除上清，将PCR管从磁力架取下，向管内加入50μL YF buffer B，用移液器轻缓吸打混匀20次；
室温孵育5min后，将PCR管置于DynaMag-96Side磁力架上3min；
移除上清，PCR管继续放置在磁力架上，向管内加入180μL 80％乙醇溶液，静置30s；
移除上清，PCR管继续放置在磁力架上，向管内加入180μL 80％乙醇溶液，静置30s后彻底移除上清；
室温静置3min，使残留乙醇彻底挥发；
将PCR管从磁力架取下，加入24μL Nuclease-free water(无核酸酶水)，移液器轻轻吸打重悬磁珠，避免产生气泡，室温静置2min；
将PCR管重新置于磁力架上，静置3min；
用移液器吸取13.5μL上清液，转移到新的200μL PCR管内，管内上清液为合并后的多重PCR产物。
3)第2轮接头序列PCR反应
采用上一步纯化后的PCR合并产物，按表10配制接头PCR反应体系(总体系30μL)：
表10.接头PCR反应体系
| 组份 | 加入量(μL) |
| 纯化后的PCR合并产物 | 13.5 |
| Enhancer buffer M | 2.5 |
| ddH<sub>2</sub>O | 2 |
| TPE1.0Index(10μM) | 1 |
| TPE2.0Index(10μM) | 1 |
| IGT-EM808polymerase mixture | 10 |
TPE1.0 Index的序列信息如下表11所示：
表11 TPE1.0 Index的序列信息
TPE2.0 Index的序列信息如下表12所示：
表12 TPE2.0 Index的序列信息
将反应混合物用漩涡振荡器震荡混匀，迷你离心机离心5秒置于PCR仪中反应。反应条件如表13中所示：
表13.接头PCR反应条件
4)第2轮磁珠纯化
向步骤3)的PCR反应产物中加入27μL室温平衡后的AMPure XP磁珠，用移液器轻缓吸打混匀20次；
室温孵育5min后，将PCR管置于DynaMag-96Side磁力架上3min；
彻底移除上清，将PCR管从磁力架取下，向管内加入50μL YF buffer B，用移液器轻缓吸打混匀20次；
室温孵育5min后，将PCR管置于DynaMag-96Side磁力架上3min；
移除上清，PCR管继续放置在磁力架上，向管内加入180μL 80％乙醇溶液，静置30s；
移除上清，PCR管继续放置在磁力架上，向管内加入180μL 80％乙醇溶液，静置30s后彻底移除上清；
室温静置3min，使残留乙醇彻底挥发；
将PCR管从磁力架取下，加入24μL Nuclease-free water，用移液器轻缓吸打混匀20次，重悬磁珠，避免产生气泡，室温静置2min；
将PCR管重新置于磁力架上，静置3min；
用移液器吸取20μL上清液，转移到新的PCR管中，管中上清为制备好的基因检测文库。
3.基因检测文库质控
取1μL步骤4)得到的基因检测文库使用Agilent 2200生物分析仪进行文库片段长度质控和浓度质控，正常文库的片段分布区间在280-420bp之间，主峰在400bp左右，无明显小片段和大段杂峰；否则建库样品不符合要求，应重新建库。
基因检测文库的长度质控和浓度质控结果如图1所示，结果显示均符合要求。
取2μL基因检测文库使用
2.0Fluorometer(ExKubit Plus dsDNA HS分析试剂盒)进行基因检测文库浓度测定，记录文库浓度。正常文库的浓度范围为5-40ng/μl，文库浓度主要与模板的质量相关。
实施例2、对30个FH患者的DNA样本进行测序分析
采用实施例1提供的家族性高胆固醇血症多重靶向捕获试剂盒，并参照现有技术中MGISEQ-2000基因测序仪使用说明书启动基因测序仪，完成DNB管、测序载片和测序试剂槽装载，并启动测序程序。
测序完成后，使用现有技术中的生物信息学软件进行数据分析，严格按照说明书操作。测序结果见表14：
表14.样本测序分析结果
| 样本编号 | 基因命名 | 碱基突变 | Chr. | Ref_Pos. | 覆盖度 |
| 1 | APOB | c.9406C>T | chr2 | 21230334 | 100％ |
| 2 | APOB | c.1379C>T | chr2 | 21252861 | 99.99％ |
| 3 | APOB | c.7331G>A | chr2 | 21232409 | 100％ |
| 4 | APOB | c.11456A>C | chr2 | 21228284 | 100％ |
| 5 | PCSK9 | c.905A>G | chr1 | 55521771 | 100％ |
| 6 | APOB | c.1837A>C | chr2 | 21250930 | 100％ |
| 7 | APOB | c.11359C>T | chr2 | 21228381 | 100％ |
| 8 | APOB | c.10167A>T | chr2 | 21229573 | 100％ |
| 9 | APOB | c.7843C>G | chr2 | 21231897 | 100％ |
| 10 | APOB | c.7729A>C | chr2 | 21232011 | 99.68％ |
| 11 | APOB | c.7724A>T | chr2 | 21232016 | 99.68％ |
| 12 | LDLR | c.694+3A>T | chr19 | 11216279 | 99.67％ |
| 13 | APOB | c.12080G>A | chr2 | 21227148 | 100％ |
| 14 | APOB | c.3708A>G | chr2 | 21237454 | 100％ |
| 15 | APOB | c.13621A>T | chr2 | 21224673 | 100％ |
| 16 | APOB | c.12614C>T | chr2 | 21225680 | 99.99％ |
| 17 | PCSK9 | c.588C>T | chr1 | 55518015 | 99.99％ |
| 18 | APOB | c.8252A>G | chr2 | 21231488 | 99.68％ |
| 19 | APOB | c.8379delC | chr2 | 21231361 | 99.67％ |
| 20 | LDLR | c.68-2A>G | chr19 | 11210897 | 99.99％ |
| 21 | APOB | c.9407G>A | chr2 | 21230333 | 100％ |
| 22 | APOB | c.8293C>T | chr2 | 21231447 | 100％ |
| 23 | APOB | c.3745C>T | chr2 | 21237417 | 99.78％ |
| 24 | APOB | c.9407G>A | chr2 | 21230333 | 100％ |
| 25 | APOB | c.5501A>G | chr2 | 21234239 | 99.45％ |
| 26 | LDLRAP1 | c.223G>A | chr1 | 25880547 | 100％ |
| 27 | LDLRAP1 | c.231+1G>T | chr1 | 25880556 | 100％ |
| 28 | PCSK9 | c.1930G>A | chr1 | 55529108 | 100％ |
| 29 | APOB | c.9452C>T | chr2 | 21230288 | 100％ |
| 30 | APOB | c.3163C>T | chr2 | 21239480 | 100％ |
结论：
本实施例构建的基因检测文库对于LDLRAP1基因、PCSK9基因、LDLR基因和APOB基因的测序覆盖度为99.45-100％，不需要使用探针捕获，利用多重PCR扩增实现待测区域的高度覆盖，具有高检测覆盖度和高均一性，样本消耗量少、样本要求低。
序列表
<110> 江苏百世诺医疗科技有限公司
<120> 一种家族性高胆固醇血症基因检测文库、其构建方法和试剂盒
<130> P210074CN-CN-BSN
<160> 384
<170> PatentIn version 3.5
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atgagggtgg gtctcaagtg a 21
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agagcctgga ttcagatggg a 21
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ctccagtgca gacttgctct 20
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tctgacccca gggcctttat 20
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ggaagaggct gatctcccac t 21
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agaggtcagg ctgaggatca c 21
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gccctcaccc aggctct 17
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ccatcacgtg tcagggcc 18
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cgctgggagg ggagga 16
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ctctgcccga tcccgg 16
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cggaaagttt ttcctgacgg agt 23
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gtgaggcccc gcctc 15
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gggcggaaag tttttcctga c 21
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tctgcccaac ttggccg 17
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tctgcccaac ttggccg 17
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acctgcactc cacttcctct 20
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atgtcttcca tggccttctt cc 22
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tgtgcttggt acccgataag tg 22
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gttgacttta tgctcattcc ctcct 25
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aggatgggga tatgggcaga g 21
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ggagcaggtc tccccaagg 19
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agcagcccca gcacctac 18
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ctctcttggg ctcctttctc tg 22
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agtggggtgg tgacttacca 20
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atttctgtgg aggtcccctc a 21
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acacacacac acacgaagga g 21
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cctttctgtg ttttcaaagc ccat 24
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aagcccagcc tcctacctg 19
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agacggagca tcccagca 18
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tcacgggcct cttcacct 18
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gggccacgct agacatgtg 19
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tcactggagc tcctgggag 19
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gtttccagct aggacacagc a 21
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tctcccaggg atggagtgat t 21
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tctgattctg gcgttgagag ac 22
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atggcgagac cctgtctcta tt 22
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tggtgttggg agacttcaca c 21
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aggtggagct gttgcactg 19
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aagcaaactg aggctcagac ac 22
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agcactcatg tctcagtccc tt 22
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gtctctagcc attggggaag ag 22
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gttcagagga tgaaactccc cc 22
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caggtgagat gagggctcct 20
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cgttttcctc ttcacgccct 20
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ccagaattcg ttgcacgcat 20
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gtctgtcctc cagcctgtg 19
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<400> 47
caggcacgtg gcactca 17
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ggacccgtct ctgggtga 18
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ggtctcgggg gcagct 16
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agctgggagg gggcc 15
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ttcgagttcc actgcctaag tg 22
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ggaaatccac ttcggcacct aa 22
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ctcatcccag tgtttaacgg gat 23
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ttccagagtt tccacaagga gg 22
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cctgtgtctc atcccagtgt 20
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cagagtttcc acaaggaggt ttc 23
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ggcctgtgtc tcatcccagt 20
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tccagagttt ccacaaggag gtt 23
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ctgtgctatg tgaaagttca attgga 26
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actgcaagat ttttcagacc aactc 25
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cttttccatc tggatcggta agga 24
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ccatcaatac attatggccc ttcg 24
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ctgaagattg tgttgatctc atcttgg 27
<210> 64
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acacttcctt tcgagttaag gaaaca 26
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tatatcccag gtttccccgg aa 22
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atcgacgtga ggttccagaa ag 22
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tgacataatc ataaaggacc cctgtg 26
<210> 68
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gaagttttag tgtactatat tgtgtaattg gagt 34
<210> 69
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ggtggagatg cctttcttgt ct 22
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ttgcacaccg tgacttcagt 20
<210> 71
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ctcataactc tcattgaaaa tatacagtat ctag 34
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ctctgtacct gctggaattg tca 23
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gatccaaagc agcaatgcca tc 22
<210> 74
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gatcttcagg ttccatcgtg ca 22
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gccattgggg tttttggtgt ac 22
<210> 76
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agtccggatt cattctgggt ct 22
<210> 77
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catggacctg aacaagagct ga 22
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cttgaattcc aagagcacac gg 22
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gtcaactgct cctttagcgg ta 22
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gaagttagcc acagctctgt ct 22
<210> 81
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cagcattggt attcagtgtg atgac 25
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gctatgtgtt cccaaaagca gtc 23
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gagctcgtcg tgagattttt cag 23
<210> 84
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gcttgaagga attcttgaaa acgac 25
<210> 85
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catttattcc tacatgggcc tcca 24
<210> 86
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ctcttttctt ttcagcccag cc 22
<210> 87
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cacatgctgg gaatcgactt g 21
<210> 88
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ctgttgaaag ctggccacat ag 22
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cactccatta ctaagctcca gtgt 24
<210> 90
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cttccatcac tgccaaagga ga 22
<210> 91
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<400> 91
ttgtgtgtga gatgtgggga ag 22
<210> 92
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<400> 92
gaccagatgc tgaacagtga g 21
<210> 93
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gtgtggaaaa cctggatggg at 22
<210> 94
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<213> Artificial Sequence
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ctgactttgc agagcaatat tctatcc 27
<210> 95
<211> 25
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<213> Artificial Sequence
<400> 95
gtctcgtgta tcttctaggg tctct 25
<210> 96
<211> 24
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<400> 96
gtgaggtgac tcagagactc aatg 24
<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 97
gcatcatcaa taaatccaac caatttctc 29
<210> 98
<211> 31
<212> DNA
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<400> 98
gtcaaacact ttgttataaa tcttattggg g 31
<210> 99
<211> 26
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<400> 99
ctaggtgctg gatgtctata ttctgt 26
<210> 100
<211> 34
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<400> 100
agctattgct aatattattg atgaaatcat tgaa 34
<210> 101
<211> 31
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<400> 101
ctgtaattct atactttttt gtgagagcag t 31
<210> 102
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 102
ccattaacct cccatttttt gagacc 26
<210> 103
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 103
tgatgggctc actgagtaaa agtg 24
<210> 104
<211> 24
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<213> Artificial Sequence
<400> 104
tcgtgtctag gaaaagcatc agtg 24
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 105
agcgagtttc ccattgccat 20
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 106
atgccatctc ttctgctgcc 20
<210> 107
<211> 23
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<213> Artificial Sequence
<400> 107
gtttcccatt gttggtgaga tcc 23
<210> 108
<211> 23
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<213> Artificial Sequence
<400> 108
cttcaaggtc agtcaagaag gac 23
<210> 109
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<213> Artificial Sequence
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tttgcattgt gttccctgaa gc 22
<210> 110
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<213> Artificial Sequence
<400> 110
cttcagcctg ctttctggat ca 22
<210> 111
<211> 26
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<213> Artificial Sequence
<400> 11
cgtagttctc atactttagg gaagca 26
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 112
gttcagagtc tcttcgttct atgct 25
<210> 113
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 113
cccaggaact agatgcatcg aa 22
<210> 114
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 114
gagattgctc tggaaatgta ctgc 24
<210> 115
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 115
tgctgtagac attcgtggag ag 22
<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 116
gatggccggg tcaaatatac ct 22
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 117
tctttcattt actttgcatg gttcaag 27
<210> 118
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 118
ttgctctttg aggctcatcc aa 22
<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 119
aatcggagag atccacaggg aa 22
<210> 120
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 120
atggctccac agtttccaag ag 22
<210> 121
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 121
tctcacttct ggcttctgct tg 22
<210> 122
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 122
tctgtggctg tttctctgaa cc 22
<210> 123
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 123
caaaatccgg aatttggact tcact 25
<210> 124
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 124
accatgttag ccagaatggt ctt 23
<210> 125
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 125
catgatgtgg aaggtgagaa aatgc 25
<210> 126
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 126
ctcaggtatt tttgttcttc accagc 26
<210> 127
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 127
ctctgttctc tctttcaaac tggcta 26
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 128
ctgaactggt ggcaaaaccc t 21
<210> 129
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 129
gcaccctgga agaaagtaat aacc 24
<210> 130
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 130
tctcccttgt tgggtctaca gat 23
<210> 131
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 131
aaaccaacgt ctggtctcat gg 22
<210> 132
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 132
ggtaaatgga ataatgctca gtgttgag 28
<210> 133
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 133
cattgagacc caaagctttc ctt 23
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 134
ctgatgccat tttcttgtct gattttc 27
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 35
cccagggact ctctgtttat gatg 24
<210> 136
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 136
catggacttc agaaaattct ctcgg 25
<210> 137
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 137
ctcagggccc tcagtggta 19
<210> 138
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 138
aggaccagga ggttcttctt ca 22
<210> 139
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 139
cccctagtac cttccaaatc cttg 24
<210> 140
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 140
tctcctgcac cctgatggg 19
<210> 141
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 141
cacagaggtg caagatgttc ct 22
<210> 142
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 142
agtggctgaa acgtgtgcat 20
<210> 143
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 143
ggccaaggtt tgaaagttca gtc 23
<210> 144
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 144
atccacatca cctccaaagc ag 22
<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 145
agccatgata ggcacatctt gag 23
<210> 146
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 146
tgcagagaca gggaaaacct tc 22
<210> 147
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 147
tgtattaata agaggatgct ccttgct 27
<210> 148
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 148
gatgttgccc gtgttgaata cat 23
<210> 149
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 149
ctcagcggac acacacaca 19
<210> 150
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 150
tcaggggcca cctctcatt 19
<210> 151
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 151
gagcaaggca caccacgat 19
<210> 152
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 152
aaggtgaaac ctgtgccgg 19
<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 153
tgagagctgt tgctggtggt 20
<210> 154
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 154
tggtccttgg tccctggtt 19
<210> 155
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 155
gtgattgctg gggacagagt g 21
<210> 156
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 156
agtgaaaaac caagaggctc tcc 23
<210> 157
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 157
gggctccccc aaacatagtt c 21
<210> 158
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 158
aagcatttga gtggagggat gg 22
<210> 159
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 159
tctgcaactc aggggagct 19
<210> 160
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 160
aaggccactg gaccctgt 18
<210> 161
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 161
ggcttcccac atgtgctca 19
<210> 162
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 162
aaggctgcca aggccac 17
<210> 163
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 163
atgtgctcag gcccctgt 18
<210> 164
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 164
agcaaggctg ccaaggc 17
<210> 165
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 165
tgcagggctt cccacatg 18
<210> 166
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 166
agagcaaggc tgccaagg 18
<210> 167
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 167
cacggcctct aggtctcct 19
<210> 168
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 168
ggaaggtggc tgtggttcc 19
<210> 169
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 169
gtttgctggg tttcttccat gtc 23
<210> 170
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 170
ccaaaaaggg tggctcacca 20
<210> 171
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 171
gtgtttgctg ctgtccaaat gg 22
<210> 172
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 172
atggattcag ctcagatggg gt 22
<210> 173
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 173
acctgctgat ttgttatagg gtgg 24
<210> 174
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 174
gccaggtcca gatggagaga 20
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 175
ctttgtgtca cagagtggga ca 22
<210> 176
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 176
tcaggcctac ttcatctctg gt 22
<210> 177
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 177
gccgggccat caccat 16
<210> 178
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 178
ccaagcccca gcctgga 17
<210> 179
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 179
ctttttgcag gttggcagct 20
<210> 180
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 180
agcactgaca cacacacaca 20
<210> 181
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 181
cgtctttgac tctaaggccc aa 22
<210> 182
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 182
cagtgggtgc ataaggagaa aga 23
<210> 183
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 183
ccaggtctgg aatgcaaagt ca 22
<210> 184
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 184
agcaaaaaca gacccagcca 20
<210> 185
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 185
gacaacacgt gtgtagtcag ga 22
<210> 186
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 186
ccattttaaa gctcagcccc ag 22
<210> 187
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 187
gacgatgcct gcctctactc 20
<210> 188
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 188
cagactctgc gatgatggag g 21
<210> 189
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 189
ctactcccca gcctcagct 19
<210> 190
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 190
actctgcgat gatggaggtt tc 22
<210> 191
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 191
cctctactcc ccagcctcag 20
<210> 192
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 192
agactctgcg atgatggagg t 21
<210> 193
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 193
ggcaacttcc gggacgat 18
<210> 194
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 194
tctgacgact ccttccaaag c 21
<210> 195
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 195
ggacgatgcc tgcctctac 19
<210> 196
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 196
gactctgcga tgatggaggt tt 22
<210> 197
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 197
tacagccgcg tcctcaac 18
<210> 198
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 198
aagccagaag ggttcggc 18
<210> 199
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 199
ggcaacttcc gggacgatg 19
<210> 200
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 200
atggaggttt cgagcccct 19
<210> 201
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 201
tcagtgggtc tttcctttga gtg 23
<210> 202
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 202
gtagagacaa agtcagacca ctcc 24
<210> 203
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 203
gactgcttgg acggctcag 19
<210> 204
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 204
accatcacag cgccagc 17
<210> 205
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 205
aggccctgct tctttttctc tg 22
<210> 206
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 206
aagcagcaag gcacagagaa t 21
<210> 207
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 207
ggtggaggtt gtaatgagcc aa 22
<210> 208
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 208
catgtcagga agcgcagagg 20
<210> 209
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 209
cgacgggtcc cctctgac 18
<210> 210
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 210
tctcacctgc gggccaa 17
<210> 211
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 211
acatctactg gaccgactct gt 22
<210> 212
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 212
aaccagttcc tgaagctcct tc 22
<210> 213
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 213
gactggcatc agcacgtga 19
<210> 214
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 214
gtgtctatcc gccacctaag tg 22
<210> 215
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 215
ctggtatagc tgatgatctc gttcc 25
<210> 216
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 216
aggagggggc agttggag 18
<210> 217
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 217
tcttggtggc cttcctttag ac 22
<210> 218
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 218
ctgttccctg tccaggagaa aa 22
<210> 219
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 219
gtgtttcctg aatgctggac tg 22
<210> 220
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 220
ctgttctgcc tcccagatga at 22
<210> 221
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 221
tggacaggtc aatcaatctt ttgga 25
<210> 222
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 222
caagttgagc aatttctgca caga 24
<210> 223
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 223
tcactgtcca gccaactata cttg 24
<210> 224
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 224
acagctgaaa gagatgaaat ttacttatct t 31
<210> 225
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 225
ctttcaacag ttccctatac atcgaga 27
<210> 226
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 226
ctcactcatt gattttctga acttccc 27
<210> 227
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 227
tgtccttcca ctcttggtag gt 22
<210> 228
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 228
acctctctga aagacaacgt gc 22
<210> 229
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 229
ccaattctcc actcgctctt gg 22
<210> 230
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 230
ctcatctgtt tttctgcttt caggg 25
<210> 231
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 231
ggtatggaga tgaagaaaat cacaatgag 29
<210> 232
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 232
cacaatgaaa acatcaacgt cctact 26
<210> 233
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 233
caagtggcat tatacacggg aga 23
<210> 234
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 234
gagataaccg tgcctgaatc tca 23
<210> 235
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 235
ggttgagggc aaatgatgaa gttc 24
<210> 236
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 236
gagacagcat cttcgtgttt caac 24
<210> 237
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 237
gtcaaggtgt gccttttctt gg 22
<210> 238
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 238
gagaacatac aagcaaagcc acc 23
<210> 239
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 239
caattttgga agtgccctgc a 21
<210> 240
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 240
ctgtctcttc ctccatggaa tttaagt 27
<210> 241
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 241
gactaccctc cacaaatttg ttgc 24
<210> 242
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 242
tcctgccatg ggcaatatta cc 22
<210> 243
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 243
gtcagaacct aggatggaga aactag 26
<210> 244
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 244
cagaaacaat gcattagatt ttgtcacc 28
<210> 245
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 245
gatttgtgat ggaatgcctg tgt 23
<210> 246
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 246
ctctgctgga aacaacgaga ac 22
<210> 247
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 247
cattgtttgt ggatgccgtg atc 23
<210> 248
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 248
ggtttatgaa tctggctccc tca 23
<210> 249
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 249
cccatttcca tgaccctttt cca 23
<210> 250
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 250
cacagtggca agtttacaca cag 23
<210> 251
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 251
aggaatgtcc tccaccttca ga 22
<210> 252
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 252
agtcccccta acagatttga gga 23
<210> 253
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 253
cagtgaaccc ttgctctacc aa 22
<210> 254
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 254
gcatctttgg ctcacatgaa gg 22
<210> 255
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 255
ttgtggtagt tccagagcct ga 22
<210> 256
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 256
gaagctaagc aatgtcctac aacaag 26
<210> 257
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 257
gctctgaagg cattgatttt ctcag 25
<210> 258
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 258
caagaaaaac tgcagcagct taaga 25
<210> 259
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 259
cggatatgat agtgctcatc aagact 26
<210> 260
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 260
gacaagtttc acatgccaag gag 23
<210> 261
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 261
ggtttctctg tacgttttcc agtac 25
<210> 262
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 262
ctctggctga cctaactcta ctaga 25
<210> 263
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 263
gggcactgac tttgtgttca ag 22
<210> 264
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 264
tgtcttccgt tctgtaatgg cc 22
<210> 265
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 265
caccctgaac cttagcaaca gt 22
<210> 266
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 266
actgttaatt tacagctaca gccct 25
<210> 267
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 267
tcagcatatg agcccatcat gtc 23
<210> 268
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 268
tggagaatga gctgaatgca gag 23
<210> 269
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 269
gtcagtgcct aagatgtcag ca 22
<210> 270
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 270
ctgatctgca aagtggcatc att 23
<210> 271
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 271
tctgacaaga caggccatat gtg 23
<210> 272
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 272
gatgggtctc tacgccacaa at 22
<210> 273
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 273
ttagagcctg ccatgaacta gc 22
<210> 274
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 274
gttgtatcaa ctgcaagtgc ctc 23
<210> 275
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 275
tgccaccaaa aggcaaagga 20
<210> 276
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 276
aggtggaggt aaaagcaaag gg 22
<210> 277
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 277
agttgaggat gtaattagca cttatatcca 30
<210> 278
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 278
cagatgaaga gaagattgaa tttgaatgga 30
<210> 279
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 279
gtctcaacac ctgcattact ttgg 24
<210> 280
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 280
tggtgccaac tgattttctt tacc 24
<210> 281
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 281
aggcctgcag tgcaggt 17
<210> 282
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 282
tccaggtgcg aagcagact 19
<210> 283
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 283
cctcatgaat tctgaacctg agact 25
<210> 284
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 284
ttgggaacat aatcaccatc cttga 25
<210> 285
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 285
ccgttttggt ggtagagacc aa 22
<210> 286
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 286
ggaaagaact cagccagaag ct 22
<210> 287
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 287
ccagctttta gggcaacatg ag 22
<210> 288
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 288
aggtgagaaa gggtggttga tc 22
<210> 289
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 289
tccagtttta ctccagcctt gg 22
<210> 290
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 290
ctctccaatg tatgggaggt tgt 23
<210> 291
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 291
aaaaccaagc tcctctccca ag 22
<210> 292
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 292
ctgttcctca cctggagcat tt 22
<210> 293
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 293
agccaaagtg gtccactaag ac 22
<210> 294
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 294
tggacatcaa gagtggggac ta 22
<210> 295
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 295
ccggagaagc atcatcaagg aa 22
<210> 296
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 296
tgctctagga aacactggca ag 22
<210> 297
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 297
gggaagtaaa aggtgtccag gaa 23
<210> 298
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 298
gacaaaccta actggggtta cca 23
<210> 299
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 299
atccctcgcc atgttgaaga tc 22
<210> 300
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 300
cagaggacac accatgagca at 22
<210> 301
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 301
cctggagagt cttcaaaaca gct 23
<210> 302
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 302
acctatggca gagccagatc ta 22
<210> 303
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 303
gagcacttga gaagtgttca gttc 24
<210> 304
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 304
gcttttaaca gaagatacag ggactg 26
<210> 305
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 305
tgaagatgag tttcaagggc ca 22
<210> 306
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 306
cttccagcaa gttgtaatgg agaag 25
<210> 307
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 307
gggaaagctg tgggctctag 20
<210> 308
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 308
ctctgcccag aattggctgt 20
<210> 309
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 309
ggtttgagag ttgtgcattt gct 23
<210> 310
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 310
caagttcctg accttcacat acca 24
<210> 311
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 311
gggtttgaga gttgtgcatt tgc 23
<210> 312
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 312
caagttcctg accttcacat accag 25
<210> 313
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 313
tcttagggtt tgagagttgt gca 23
<210> 314
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 314
gttcctgacc ttcacatacc aga 23
<210> 315
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 315
gctgggtcaa gtgatggaag ag 22
<210> 316
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 316
ttggggtgca agagtcaaag g 21
<210> 317
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 317
tattttggct gaggctgggt ca 22
<210> 318
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 318
ttggggtgca agagtcaaag ga 22
<210> 319
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 319
ctgggtcaag tgatggaaga ga 22
<210> 320
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 320
cttggggtgc aagagtcaaa g 21
<210> 321
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 321
gccctccctc tggcctag 18
<210> 322
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 322
tgaggagccc gcccag 16
<210> 323
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 323
gcttcccaca tgtgctcagg 20
<210> 324
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 324
tcccacacca gacagccc 18
<210> 325
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 325
tcctgcctgg tacacaatag gt 22
<210> 326
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 326
accttaccaa ctagctgctc ct 22
<210> 327
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 327
cctcagcaat tcctgcaaag g 21
<210> 328
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 328
aggacagggt accacacaga a 21
<210> 329
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 329
gcgatgctat ccgatgaaca ga 22
<210> 330
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 330
gcttctctac ttcccagcaa ca 22
<210> 331
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 331
ttccctttgg aggattgctc ac 22
<210> 332
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 332
tccatattcg gcagactctc ca 22
<210> 333
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 333
ccggggatga tgatgattgc aa 22
<210> 334
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 334
cacaccagct ctcagaagtc t 21
<210> 335
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 335
gctgtccaag gagctgca 18
<210> 336
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 336
ggatggcgct gaggcc 16
<210> 337
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 337
aagttcatac ctcagcggac ac 22
<210> 338
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 338
tctgcagctt catcctgaag ac 22
<210> 339
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 339
caaggatcca tattcccaca gct 23
<210> 340
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 340
catcacctta ttcaatcctc actgc 25
<210> 341
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 341
caaagaactg tgtgtgggag ga 22
<210> 342
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 342
gcatttgaat gtccccgtgt atg 23
<210> 343
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 343
tactaccccc agaacatcac ca 22
<210> 344
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 344
cagatcacaa tgaggggctg at 22
<210> 345
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 345
cagtctatgg ccagagtgat catg 24
<210> 346
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 346
gaagctgcct cccaatgttt tg 22
<210> 347
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 347
ggtttgagag ttgtgcattt gct 23
<210> 348
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 348
caagttcctg accttcacat acca 24
<210> 349
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 349
gggtttgaga gttgtgcatt tgc 23
<210> 350
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 350
caagttcctg accttcacat accag 25
<210> 351
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 351
tcttagggtt tgagagttgt gca 23
<210> 352
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 352
gttcctgacc ttcacatacc aga 23
<210> 353
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 353
tcttagggtt tgagagttgt gcat 24
<210> 354
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 354
gttcctgacc ttcacatacc agaa 24
<210> 355
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 355
ggtttgagag ttgtgcattt gctt 24
<210> 356
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 356
ccttcacata ccagaattcc agct 24
<210> 357
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 357
gctgggtcaa gtgatggaag ag 22
<210> 358
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 358
ttggggtgca agagtcaaag g 21
<210> 359
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 359
tattttggct gaggctgggt ca 22
<210> 360
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 360
ttggggtgca agagtcaaag ga 22
<210> 361
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 361
ctgggtcaag tgatggaaga ga 22
<210> 362
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 362
cttggggtgc aagagtcaaa g 21
<210> 363
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 363
ggtcaagtga tggaagagaa acaga 25
<210> 364
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 364
agacccaaag acttagggtc aatg 24
<210> 365
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 365
gggtcaagtg atggaagaga aacag 25
<210> 366
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 366
cagatctaga cccaaagact taggg 25
<210> 367
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 367
cctctactcc ccagcctcag 20
<210> 368
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 368
agactctgcg atgatggagg t 21
<210> 369
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 369
gcctgcctct actcccca 18
<210> 370
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 370
tgctattcag actctgcgat gatg 24
<210> 371
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 371
cctgcctcta ctccccagc 19
<210> 372
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 372
tgctattcag actctgcgat ga 22
<210> 373
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 373
gcctgcctct actccccag 19
<210> 374
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 374
tgctattcag actctgcgat gat 23
<210> 375
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 375
gacgatgcct gcctctactc 20
<210> 376
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 376
cactgctatt cagactctgc ga 22
<210> 377
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 377
ggcaacttcc gggacgat 18
<210> 378
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 378
actgctattc agactctgcg atg 23
<210> 379
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 379
ggacgatgcc tgcctctac 19
<210> 380
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 380
cactgctatt cagactctgc gat 23
<210> 381
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 381
gcaacttccg ggacgatgc 19
<210> 382
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 382
cactgctatt cagactctgc g 21
<210> 383
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 383
ttgtcttcct aaggaacgac atggctacga tccgactt 38
<210> 384
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 384
agtcggaggc caagcggtct taggaagaca a 31
Claims (1)
1.一种用于DNBSEQ测序平台的家族性高胆固醇血症基因检测文库的构建方法，其特征在于，包括如下步骤：
1)获得DNA样本；
2)采用引物组1、引物组2、引物组3分别对所述DNA样本进行第1轮多重PCR反应得到扩增产物；
3)第1轮多重PCR反应得到的PCR产物先分别加入接头引物1和接头引物2进行连接反应，反应完毕后的PCR产物以体积比6-8﹕5-7﹕1-3合并，然后PCR合并产物进行第2轮接头序列PCR反应，得到基因检测文库；
所述引物组1的序列为SEQ ID NO.1-152，检测如下基因：LDLRAP1基因、LDLR基因，APOB基因，PCSK9基因；
所述引物组2的序列为SEQ ID NO.153-322，检测如下基因：LDLR基因，APOB基因，PCSK9基因，LDLRAP1基因；
所述引物组3的序列为SEQ ID NO.323-382，检测如下基因或基因位点：LDLRAP1基因，CELSR2基因的多态性位点rs629301，ABCG8基因的多态性位点rs4299376，SLC22A1基因的多态性位点rs1564348，HFE基因的多态性位点rs1800562，MYLIP基因的多态性位点rs3757354，ST3GAL4基因的多态性位点rs11220462，NYNRIN基因的多态性位点rs8017377，APOE基因的多态性位点rs429358、rs7412，LDLR基因的多态性位点rs6511720，APOB基因的多态性位点rs1367117，PCSK9基因的多态性位点rs2479409；
所述接头引物1的序列为SEQ ID NO.383所示，所述接头引物2的序列为SEQ ID NO.384所示；
其中，引物组1和引物组2的第1轮多重PCR反应的反应条件如下：
引物组3的第1轮多重PCR反应的反应条件如下：
第2轮接头序列PCR反应的反应条件如下：
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| CN109576799A (zh) * | 2018-11-30 | 2019-04-05 | 安吉康尔（深圳）科技有限公司 | Fh测序文库的构建方法和引物组及试剂盒 |
| CN111808855A (zh) * | 2020-07-20 | 2020-10-23 | 北京安智因生物技术有限公司 | 一种遗传性家族性高胆固醇血症的通用基因检测文库的构建方法及其试剂盒 |
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| CN109576799A (zh) * | 2018-11-30 | 2019-04-05 | 安吉康尔（深圳）科技有限公司 | Fh测序文库的构建方法和引物组及试剂盒 |
| CN111808855A (zh) * | 2020-07-20 | 2020-10-23 | 北京安智因生物技术有限公司 | 一种遗传性家族性高胆固醇血症的通用基因检测文库的构建方法及其试剂盒 |
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