CN105861547A - 身份证号码永久嵌入基因组的方法 - Google Patents

身份证号码永久嵌入基因组的方法 Download PDF

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CN105861547A
CN105861547A CN201610133919.7A CN201610133919A CN105861547A CN 105861547 A CN105861547 A CN 105861547A CN 201610133919 A CN201610133919 A CN 201610133919A CN 105861547 A CN105861547 A CN 105861547A
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Abstract

本发明提供了身份证号码永久嵌入基因组的方法，其一是把含有身份证号码的外源性基因片段，通过CRISPR技术，永久的插入到基因组中，其二是将含有身份证号码的特定DNA分离出来读取。这样使得永久保存的DNA一直有办法鉴定身份，即使是外面的包装标签遗失或弄错了。其可以同样用于微生物(包括细菌和病毒)的微生物保存。比如说SARS病毒，如果能留下一个失去活性的SARS病毒基因标本，就可以供后人研究。

Description

身份证号码永久嵌入基因组的方法
技术领域
本发明属于基因提取、运输和保存领域，具体涉及身份证号码永久嵌入基因组的方法。
背景技术
目前的基因提取、运输和保存主要是依靠试管上的标识，由于环节多，试管外面写的标识容易弄错或磨损。对于国家基因库的建立，确保基因的身份永远不被弄错是至关重要的。所以，我们要在基因上面永久性的贴上用DNA编辑的身份证号码。这样，即使试管没有任何标签，只要样本在，身份证号码就在。
将数据信息转化为DNA信息永久保存，目前有两种方法。一种是不将基因序列直接插入基因组中，而是将化学物质与基因组样本混合，利用化学物质的结构来代表数据信息，而此种方法的缺点在于解析数据信息时，不仅要对化学物质检测，还要对基因组样本检测，这样就比较麻烦。
另外一种是不用CRISPR-Cas9方法，而是直接利用限制酶剪切的方法切开基因组再将代表身份证号码的序列连接到基因组上，此种方法的缺点是限制酶的碱基只有4-6个，这样不能保证靶向的精准性。
发明内容
将数据信息转化为DNA信息永久保存，目前主要还是一个设想。比如说，瑞士苏黎世大学的Robert Grass教授在2015年发表文章指出，从理论上来说，1克DNA可以存下整个互联网的信息。但目前还没有人尝试一个相对简单而又非常实用的事情，如将中国的身份证号码转换成DNA保存。同时，这两年发展起来的基因编辑技术(CRISPR)，可以让研究人员非常容易的在基因组中插入新的片段。CRISPR(Clustered Regularly InterspersedShort Palindromic Repeats，规律间隔成簇短回文重复序列)是细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统。科学家们利用RNA引导Cas9核酸酶可在多种细胞(包括iPS)的特定的基因组位点上进行切割。
本发明旨在发明便于检测及靶向精准性高的一种身份证号码永久嵌入基因组的方法。
为了解决上述问题，本发明提供了身份证号码永久嵌入基因组的方法，包括：将身份证上用到的数字0-9和字母X分别转换成各自不同的含有两个碱基的碱基对，将身份证号码按照数字和/或字母按照次序转化为含有上述碱基对的DNA序列的设计身份证序列；在上述DNA序列的两条单链上的两端分别加上限制酶的酶切位点及在每条单链其中一端的限制酶的酶切位点的外端加上与DNA插入位点互补的碱基序列；通过CRISPR-Cas9方法将目的基因组中的目的位点切开及通过人工合成生成合成身份证序列；将人工合成的身份证序列、T4DNA连接酶和第二缓冲液与切开后的基因组混合并在适当温度下反应适当时间，使身份证序列连接到目的位点上。
根据上述身份证号码永久嵌入基因组的方法，还包括：当需要进行身份证号码核对的时候，用所述限制酶进行切割将身份证序列与基因组分离开，然后纯化并测序以确定对应的身份证号码。
根据上述身份证号码永久嵌入基因组的方法，其中，CRISPR-Cas9方法包括：根据目的序列人工合成sgRNA，在sgRNA引导下及在第一缓冲液中利用Cas9对目的基因组进行切割。
根据上述身份证号码永久嵌入基因组的方法，其中，Cas9是一种具有切割双链DNA能力的核酸酶。
根据上述身份证号码永久嵌入基因组的方法，其中，选择的目的序列必须含有一个PAM序列。
根据上述身份证号码永久嵌入基因组的方法，其中，PAM序列为5’-NGG-3’，其中N选自A、T、C或G。
根据上述身份证号码永久嵌入基因组的方法的聚光发电方法，其中，5’-NGG-3’的5’端应该带有20bp左右的基因组互补序列以保证sgRNA的靶向的精准性。
根据上述身份证号码永久嵌入基因组的方法，其中，限制酶为限制性核酸内切酶，能够识别特定的核苷酸序列，并在每条链中特定部位的两个核苷酸之间的磷酸二酯键进行切割。
根据上述身份证号码永久嵌入基因组的方法，其中，限制酶包括BamHI和EcoRI。
根据上述身份证号码永久嵌入基因组的方法的聚光发电方法，其中，在适当温度下反应适当时间为在4℃下反应过夜或在16℃下反应4小时左右。
有益效果
本发明公开的身份证号码永久嵌入基因组的方法，其一是把含有身份证号码的外源性基因片段，通过CRISPR技术，永久的插入到基因组中，其二是将含有身份证号码的特定DNA分离出来读取。这样使得永久保存的DNA一直有办法鉴定身份，即使是外面的包装标签遗失或弄错了。其可以同样用于微生物(包括细菌和病毒)的微生物保存。比如说SARS病毒，如果能留下一个失去活性的SARS病毒基因标本，就可以供后人研究。
附图说明
图1是加上限制酶的酶切位点和与DNA插入位点互补的碱基序列后的两条身份证序列的示意图；
图2是用CRISPR-Cas9系统切割目标DNA序列示意图；
图3是利用T4DNA连接酶将合成身份证序列插入到基因组上的示意图；
图4是用限制酶将身份证序列分离出来的示意图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细描述，但不作为对本发明的限定。
首先人工合成代表身份证号码的DNA序列，即将身份证上用到的数字0-9和字母X转换成含有两个碱基的DNA序列，其由选自A、T、C和G四种碱基任意组合，其中两个碱基也可以选择相同的其中一个碱基。这样，18位数的身份证就需要36个碱基来表示。下面我们列举了其中一种表示方法：如表1所示：
表1
0->AA 1->AC 2->AG 3->AT 4->CA 5->CC
6->CG 7->CT 8->GA 9->GC X->GG
图1是加上限制酶的酶切位点和与DNA插入位点互补的碱基序列后的两条身份证序列的示意图，如图1所示，然后，我们在上述DNA序列的两条单链(图1中所示的身份证序列和身份证序列的互补序列)上的两端分别加上限制酶的酶切位点及在每条单链其中一端的限制酶的酶切位点的外端加上与DNA插入位点互补的碱基序列(图1中所示的互补序列)。其中，两条单链上加上与DNA插入位点互补的碱基序列的一端彼此相反。
假如我们要表示的身份证号是110425199601013838，用BamHI和EcoRI作为作为限制酶切割，那么加上限制酶的酶切位点和与DNA插入位点互补的碱基序列后的两条身份证序列应该是：
3’->5’(N代表基因组的互补序列)
GGATCCACACAACAAGCCACGCGCCCAAACAAACATGAATGACTTAAGNNNNN
5’->3’(N代表基因组的互补序列)
NNNNNCCTAGGTGTGTTGTTCGGTGCGCGGCTTTGTTTGTACTTACTGAATTC
设计出身份证序列后，通过人工合成生成相应的身份证序列。
然后，我们通过CRISPR-Cas9(Clustered Regularly Interspaced ShortPalindromic Repeats-Cas9)技术将基因组中的目的位点切开。
CRISPR-Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA，防御机制简单地说就是在特定RNA的引导下，核酸酶Cas9切割病毒或者外源DNA从而使之降解或失活。而通过人工设计RNA，可以形成具有引导作用的sgRNA(单链引导RNA)，足以引导Cas9对DNA的定点切割。
Cas9是一种可以切割双链DNA的核酸酶，但需要有sgRNA(single guide RNA，单链引导RNA)引导才能对目的基因进行切割，因此先根据目的序列人工合成sgRNA，选择的目的序列必须含有一个PAM序列，双链复合体结构从此处开始形成。所谓的PAM序列即为5’-NGG-3’(N代表A、T、C、G任意一种)，理论上每8个碱基就能找到一个，因此对基因组任意一处都可以进行编辑，这也是其它基因编辑技术无法比拟的优点。为了保证靶向的精准性，sgRNA的5’端应该带有20bp左右的基因组互补序列。
假如我们要对基因组某一处进行编辑且已经知道到了该处的碱基序列，那么可以设计如下一个sgRNA：
3’->5’(N代表基因组的互补序列)
UUUUCGUGGGCUGAGCCACGGUGAAAAAGUUCAACUAUUGGCCUGAUCGGAAUAAAAUUUCGAUAAACAUCGAGAUUUUGNNNNNNNNNNNNNNNNNNNN
将Cas9、sgRNA、第一缓冲液与基因组混合，在适当温度反应适当时间。第一缓冲液选择能够保证Cas9和sgRNA的酶活性的缓冲液，Cas9从生物公司购买，各组分含量与反应条件参照说明书即可，图2是用CRISPR-Cas9系统切割目标DNA序列示意图，过程如图2所示。
最后加入人工合成的身份证序列、T4DNA连接酶和第二缓冲液并在适当温度下反应适当时间，让身份证序列连接到目的位点上。第二缓冲液选择能够保证T4DNA连接酶的酶活性的缓冲液，T4DNA连接酶从生物公司购买，各组分含量与反应条件参照说明书即可，图3是利用T4DNA连接酶将合成身份证序列插入到基因组上的示意图，具体过程如图3所示。
当需要进行身份证号码核对的时候，用前述设计的限制酶进行切割将身份证序列与基因组分离开，然后纯化并测序就可以知道对应的身份证号码了，纯化步骤可以采用小片段DNA的琼脂糖凝胶回收，使用DNA切胶回收试剂盒即可，具体操作根据试剂盒的不同而不同。图4是用限制酶将身份证序列分离出来的示意图，具体过程如图4所示。
如要分离前述实施例中设计的身份证序列的话，那么需要使用BamHI和EcoRI两种限制酶进行切割，便会得到如下结果：
3’->5’(N代表基因组的互补序列)
NNNNNG GATCCACACAACAAGCCACGCGCCGAAACAAACATGAATGACTTAA GNNNNN
NNNNNCCTAG GTGTGTTGTTCGGTGCGCGGCTTTGTTTGTACTTACTG AATTCNNNNN
5’->3’(N代表基因组的互补序列)
将片段纯化即得到：
3’ GATCCACACAACAAGCCACGCGCCGAAACAAACATGAATGACTTAA 5’
5’ GTGTGTTGTTCGGTGCFCGGCTTTGTTTGTACTTACTG 3’
再对此片段进行测序即可得到对应的身份证号码：110425199601013838。
以上所述，仅是本发明较佳的实施方式，并非对本发明的技术方案做任何形式上的限制。凡是依据本发明的技术实质对以上实施例做任何简单修改，形式变化和修饰，均落入本发明的保护范围。

Claims (10)

1.身份证号码永久嵌入基因组的方法，其特征在于：所述方法包括：
将身份证上用到的数字0-9和字母X分别转换成各自不同的含有两个碱基的碱基对，将身份证号码按照数字和/或字母按照次序转化为含有上述碱基对的DNA序列的设计身份证序列；
在上述DNA序列的两条单链上的两端分别加上限制酶的酶切位点及在每条单链其中一端的限制酶的酶切位点的外端加上与DNA插入位点互补的碱基序列及通过人工合成生成合成身份证序列；
通过CRISPR-Cas9方法将目的基因组中的目的位点切开；
将人工合成的身份证序列、T4 DNA连接酶和第二缓冲液与切开后的基因组混合并在适当温度下反应适当时间，使身份证序列连接到目的位点上。
2.根据权利要求1所述的身份证号码永久嵌入基因组的方法，其特征在于：所述方法包括：当需要进行身份证号码核对的时候，用所述限制酶进行切割将身份证序列与基因组分离开，然后纯化并测序以确定对应的身份证号码。
3.根据权利要求1所述的身份证号码永久嵌入基因组的方法，其特征在于：所述CRISPR-Cas9方法包括：根据目的序列人工合成sgRNA，在sgRNA引导下及在第一缓冲液中利用Cas9对目的基因组进行切割。
4.根据权利要求3所述的身份证号码永久嵌入基因组的方法，其特征在于：所述Cas9是一种具有切割双链DNA能力的核酸酶。
5.根据权利要求3所述的身份证号码永久嵌入基因组的方法，其特征在于：选择的所述目的序列必须含有一个PAM序列。
6.根据权利要求5所述的身份证号码永久嵌入基因组的方法，其特征在于：所述PAM序列为5’-NGG-3’，其中N选自A、T、C或G。
7.根据权利要求5所述的身份证号码永久嵌入基因组的方法，其特征在于：所述5’-NGG-3’的5’端应该带有20bp左右的基因组互补序列以保证sgRNA的靶向的精准性。
8.根据权利要求1所述的身份证号码永久嵌入基因组的方法，其特征在于：所述限制酶为限制性核酸内切酶，能够识别特定的核苷酸序列，并在每条链中特定部位的两个核苷酸之间的磷酸二酯键进行切割。
9.根据权利要求8所述的身份证号码永久嵌入基因组的方法，其特征在于：所述限制酶包括BamHI和EcoRI。
10.根据权利要求1所述的身份证号码永久嵌入基因组的方法，其特征在于：在所述适当温度下反应所述适当时间为在4℃下反应过夜或在16℃下反应4小时左右。
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