CN105177126A - 一种利用荧光pcr技术对小鼠的分型鉴定方法 - Google Patents
一种利用荧光pcr技术对小鼠的分型鉴定方法 Download PDFInfo
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Abstract
本发明涉及一种利用荧光PCR技术对小鼠的分型鉴定方法，利用荧光PCR引物，经PCR扩增后将产物与已知片段大小的ROX混合，利用377测序，使用Genemapper软件计算出PCR产物的大小，从而达到鉴定小鼠基因型的目的，即可。本发明简单直接准确的鉴定出Crispr/Cas9敲除的mircoRNA小鼠。
Description
技术领域
本发明属于小鼠分型鉴定方法领域，特别涉及一种利用荧光PCR技术对小鼠的分型鉴定方法。
背景技术
基因打靶技术自诞生以来一直是研究基因功能的重要手段之一，揭示了许多重要基因的生物学功能。除此之外，研究人员也憧憬能利用基因打靶技术对特定基因进行敲除或者修饰，从而达到治疗疾病或者改善畜禽生产性状的目的。但早期基因打靶技术效率极低，难以真正应用到医疗或者畜禽改良实践中。CRISPR/Cas9系统作为一种新兴的基因定点编辑技术逐渐成熟并在多个动植物物种中成功得到应用,极大地促进了基因功能的研究。CRISPR/Cas系统广泛分布于细菌和古生菌基因组中,是在进化过程中形成的一种适应性免疫系统,可以降解入侵病毒或质粒DNA。
CRISPR/Cas系统的出现为基因工程提供了一个强有力的应用新工具,它将给基因组定向编辑的研究和应用领域带来突破性的技术革命,特别是在基因功能解析、人类疾病靶向治疗等应用中有巨大的潜力和广阔的前景；有望加速重要农作物水稻、小麦性状改良与分子定向育种。更令人鼓舞的是,其操作简单、实验周期短、节约成本，有利于在普通实验室推广这一技术,因此,CRISPR/Cas系统的广泛应用将对生物学研究产生深远的影响。利用Crispr/Cas9技术的基因敲除小鼠也越来越多，传统的对敲除小鼠进行的分型的方法是通过T7E1酶切来鉴定。这种方法首先通过PCR技术扩增出大量的需要鉴定位点的模板，再通过一个退火的过程会在杂合链上形成一个缺口，最后使用T7E1酶切并观察琼脂糖电泳PCR产物条带来鉴定小鼠的基因型。然而，这种方法过于繁琐，需要经过一段时间退火才可以进行下一步实验；使用酶切方法，如果酶切的效率低也会产生假阴性的结果；使用琼脂糖电泳，分辨率较低。
发明内容
本发明所要解决的技术问题是提供一种利用荧光PCR技术对小鼠的分型鉴定方法，经过PCR扩增后通过产物大小及峰图直观分辨小鼠类型。
本发明的一种利用荧光PCR技术对小鼠的分型鉴定方法，包括：
(1)小鼠饲养和样本采集：
SPF级C57BL/6J小鼠(上海斯莱克实验动物有限公司)，SPF级利用Crispr/Cas9系统敲除mircoRNA的C57BL/6J小鼠(中科院实验动物中心构建)，得到的小鼠为Founder小鼠；
将2只Founder小鼠与C57BL/6J小鼠杂交，得到F1代小鼠，F1代杂合小鼠得到F2代；
(2)DNA抽取：取上述F1代小鼠1cm尾组织(－20℃保存备用)，然后提取DNA；
采用动物基因组DNA抽提试剂盒(生工生物工程有限公司)，按照说明书进行抽提得到DNA，以0.8％琼脂糖凝胶电泳确定DNA质量和浓度；
(3)引物设计：
在Crispr/Cas9系统敲除的基因位点区域设计一对PCR引物(根据NCBI数据库中C57BL/6J小鼠的microRNA的序列，使用Primer3在线软件(http://frodo.wi.mit.edu/primer3/)进行初步引物设计，再利用oligo6人工设计，然后合成(上海生工生物工程技术服务有限公司))，且在上游引物的5’端使用FAM荧光修饰，得到荧光引物；
(4)荧光PCR技术鉴定：
将荧光引物对步骤(2)中的DNA进行PCR扩增(对F1和F2代DNA进行相同条件的扩增)，然后将PCR产物与分子内标(携带ROX的已知片段大小的DNA)混合，在测序仪上经过电泳分离，通过使用分子量内标法，进行鉴定。
其中鉴定过程中使用Genemapper软件计算出PCR产物大小，从而达到鉴定小鼠基因型的目的。
所述步骤(1)中mircoRNA为mircoRNA505。
所述步骤(3)中引物为：L：AAACCAGCAAGTGTTGACGC；R：CCCTGTTTGTCACTTGCAGA。
所述步骤(3)中FAM为6’FAM。
所述步骤(4)中PCR扩增具体为：3μLDNA加入到Taq酶体系中，其中Taq酶体系包含1.5μL200nM上下游引物、1.5μL0.25mM的dNTP，5μL10XPCRBuffer，1μL的1单位Taq酶，并使用矿物油覆盖；同时设阴性对照，反应程序为：95℃变性5min；94℃变性30s，56℃复性1min30s，72℃延伸1min，35个循环，72℃，10min。
所述步骤(4)中在测序仪上经过电泳分离为：在377测序仪上经过聚丙烯酰胺凝胶电泳分离，其中测试仪功率为30W，分离时间为2h。
所述步骤(4)中分子内标为携带ROX的已知片段大小的DNA，其中已知片段大小的DNA的片段大小为79、105、131、151。
步骤(4)中鉴定具体：使用GeneScanTM672将测序仪上电泳分离的结果进行数据采集，然后使用Genemapper软件对采集数据进行分析，分析PCR产物检测峰的数目及位置，计算出PCR产物大小，进行鉴定。
检测峰的数目为单峰代表纯合，双峰代表杂合即为单敲小鼠，再根据检测峰的位置鉴定野生和双敲小鼠。
鉴定双敲(单峰，峰的位置居于左侧，表示产物长度较小)，单敲(双峰，左侧峰与双敲小鼠一致，右侧峰与野生型小鼠一致)，野生型(单峰，峰的位置居于右侧，表示产物长度较长)小鼠。
不同位置代表不同PCR产物大小，所述野生老鼠产物大小为112bp；双敲小鼠产物大小为89bp；单敲小鼠产物大小为89bp/112bp(如图1)。
通过使用分子量内标法具体指：利用携带ROX(红色荧光染料)的已知片段大小DNA的四种DNA混合物作为内参，四种DNA片段大小分别为79、105、131、151。
本发明利用荧光PCR技术方法可以简洁、方便、准确地鉴定Crispr/Cas9系统敲除mircoRNA的小鼠。利用引物设计软件在敲除位点区域设计一对PCR引物，并且在上游引物的5’端使用FAM荧光修饰。在经过PCR的扩增后，将PCR产物与已知片段大小的ROX混合，在377测序仪上经过聚丙烯酰胺凝胶电泳分离。通过已知片段大小的ROX使用分子量内标法，使用Genemapper软件计算出PCR产物大小，从而达到鉴定小鼠基因型的目的。其分辨率远远大于琼脂糖电泳。
有益效果
本发明利用荧光PCR技术准确直接地鉴定出Crispr/Cas9系统敲除mircoRNA的小鼠，使用一对在上游引物5’端用FAM荧光修饰的在敲除位点区域特异性的荧光引物，经PCR扩增后通过产物大小及峰图直观的分辨出小鼠类型。而传统的对敲除小鼠进行的分型的方法是通过T7E1酶切来鉴定，这种方法首先通过PCR技术扩增出大量的需要鉴定位点的模板，再通过一个退火的过程会在杂合链上形成一个缺口，最后使用T7E1酶切并观察琼脂糖电泳PCR产物条带来鉴定小鼠的基因型。然而，这种方法过于繁琐，需要经过一段时间退火才可以进行下一步实验；使用酶切方法，如果酶切的效率低也会产生假阴性的结果；使用琼脂糖电泳，分辨率较低。
附图说明
图1为荧光PCR对敲除小鼠鉴定结果图；其中A为野生型小鼠、B为双敲小鼠、C为单敲小鼠；
图2为Founder小鼠部分基因组测序图(敲除部分测序序列)，其中有套峰存在，说明利用Crispr/Cas9系统敲除C57BL/6J小鼠的microRNA-505基因敲除成功，被敲除了23bp。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
(1)小鼠饲养和样本采集：将经鉴定过的Founder小鼠(本人使用Crispr/Cas9系统敲除mircoRNA505(GeneID:751545)的C57BL/6J小鼠作为Founder小鼠，经鉴定该Founder小鼠敲除23bp，记为Founder17)与C57BL/6J小鼠杂交产生F1代小鼠。取F1代小鼠1cm尾组织，－20℃保存备用。
(2)DNA抽提：将(1)中尾组织采用生工生物工程有限公司的动物基因组DNA抽提试剂盒，按照说明书进行抽提得到DNA，以0.8％琼脂糖凝胶电泳确定DNA质量和浓度。
(3)引物设计：在Crispr/Cas9系统敲除的基因位点区域设计一对PCR引物，根据NCBI数据库中C57BL/6J小鼠的microRNA505的序列，使用Primer3在线软件(http://frodo.wi.mit.edu/primer3/)进行初步引物设计，再利用oligo6人工设计，并由上海生工生物工程技术服务有限公司合成，且在上游引物的5’端使用FAM荧光修饰，下表即为所设计的引物。
Table1.荧光PCR引物
注：505-377-L引物5’端使用6’FAM修饰。
荧光PCR技术鉴定：将50ng的F1代小鼠尾巴DNA3μL加入到Taq酶体系中，其中Taq酶体系包含1.5μL200nM上下游引物、1.5μL0.25mM的dNTP，5μL10XPCRBuffer，1μL的1单位Taq酶，并使用矿物油覆盖；同时设阴性对照。PCR反应程序为:95℃变性5min；94℃变性30s，56℃复性1min30s，72℃延伸1min，35个循环，72℃10min，经过PCR扩增后，将PCR产物稀释5倍后等体积地与ROX混合，吸取1ul上样到377测序仪上，功率为30W，电泳时间为2h；
使用GeneScanTM672(AppliedBiosystems)软件将377测序仪上电泳分离的结果进行数据采集，然后使用Genemapper软件((AppliedBiosystems))对采集数据进行分析，分析得到的PCR产物检测峰的数目及位置，计算出PCR产物大小，进行鉴定。
图1即为分析结果，通过检测峰的数目以及位置可以轻易分辨野生，双敲，单敲的老鼠(野生老鼠产物大小为112bp，单峰，图1中A；双敲小鼠产物大小为89bp，单峰，图1中B；单敲小鼠产物大小为89bp/112bp，双峰，图1中C)。
Claims (10)
1.一种利用荧光PCR技术对小鼠的分型鉴定方法，包括：
(1)利用Crispr/Cas9系统敲除mircoRNA的C57BL/6J小鼠，得到Founder小鼠；将Founder小鼠与C57BL/6J小鼠杂交，得到F1代小鼠，F1代杂合小鼠得到F2代；
(2)分别取上述F1、F2代小鼠尾组织，然后提取DNA；
(3)在Crispr/Cas9系统敲除的基因位点区域设计一对PCR引物，且在上游引物的5’端使用FAM荧光修饰，得到荧光引物；
(4)将荧光引物对步骤(2)中的DNA进行PCR扩增，然后将PCR产物与分子内标混合，在测序仪上经过电泳分离，鉴定。
2.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(1)中mircoRNA为mircoRNA505。
3.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(3)中引物为：L：5’AAACCAGCAAGTGTTGACGC3’；R：5’CCCTGTTTGTCACTTGCAGA3'。
4.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(3)中FAM为6’FAM。
5.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(4)中PCR扩增具体为：3μLDNA加入到Taq酶体系中，其中Taq酶体系包含1.5μL200nM上下游引物、1.5μL0.25mM的dNTP，5μL10XPCRBuffer，1μL的1单位Taq酶，并使用矿物油覆盖；同时设阴性对照，反应程序为：95℃变性5min；94℃变性30s，56℃复性1min30s，72℃延伸1min，35个循环，72℃，10min。
6.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(4)中在测序仪上经过电泳分离为：在377测序仪上经过聚丙烯酰胺凝胶电泳分离，其中测试仪功率为30W，分离时间为2h。
7.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(4)中分子内标为携带ROX的已知片段大小的DNA，其中已知片段大小的DNA的片段大小为79、105、131、151。
8.根据权利要求1所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：所述步骤(4)鉴定具体为：将测序仪上电泳分离的结果进行数据采集，然后对采集的数据进行分析，分析PCR产物检测峰的数目及位置，计算出PCR产物大小，进行鉴定。
9.根据权利要求8所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：检测峰的数目：单峰为纯合，双峰为杂合；检测峰的位置：峰的位置代表产物片段大小。
10.根据权利要求8所述的一种利用荧光PCR技术对小鼠的分型鉴定方法，其特征在于：野生老鼠产物大小为112bp，检测峰位于右侧，为单峰；双敲小鼠产物大小为89bp检测峰位于左侧，为单峰；单敲小鼠产物大小为89bp，左侧峰，112bp为右侧峰，为双峰。
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| CN103710372B (zh) * | 2013-12-31 | 2015-08-12 | 东华大学 | 一种用于功能研究的miR-505高表达载体的构建方法 |
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| CN104212836A (zh) * | 2014-09-18 | 2014-12-17 | 东华大学 | 一种在哺乳动物细胞系中敲除mir-505的方法 |
| CN104651401B (zh) * | 2015-03-05 | 2019-03-08 | 东华大学 | 一种mir-505双等位基因敲除的方法 |
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