CN107034210A - 增强子筛选高通量测序文库简易构建的载体制作方法 - Google Patents
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Abstract
本发明公开了增强子筛选高通量测序文库简易构建的载体制作方法,包括以下步骤:步骤一,构建含有SpeI单酶切位点和EcoRI酶切位点质粒1;步骤二,使用多重PCR扩增待测增强子群;步骤三,连接高通量测序接头序列1和2,使用引物1和引物2进行PCR扩增,构建含有待测增强子的DNA文库1;步骤四,使用引物3和引物4,PCR扩增DNA文库1,得到DNA文库2;步骤五,使用SpeI和EcoRI双酶切质粒1和DNA文库2,酶联两者的双酶切产物,构建成结构含有多个待测增强子的质粒群2;本发明的设计使得对其所含待测增强子的转录本RNA进行高通量测序文库构建的步骤由常规步骤变成了引物2反转录和PCR两步。此外,使用引物2反转录,避免了oligo dT反转录过程中将大量无关的mRNA转录,降低了后续测序成本和数据分析难度。
Description
技术领域
本发明涉及生物技术领域,具体为增强子筛选高通量测序文库简易构建的载体制作方法。
背景技术
现有方法通过RNA高通量测序来筛选潜在的增强子,需要使用Oligo dT将RNA反转录成cDNA,然后再按照DNA文库构建过程进行文库构建,步骤包括末端修复、链接测序接头、PCR等步骤。此外,使用Oligo dT来进行反转录,将细胞内与目标RNA不相关的所有其他mRNA也进行了反转录,使得后续测序中产生了许多不相关的测序结果,提高了测序成本,也增加了后续数据分析难度。
发明内容
本发明的目的在于提供增强子筛选高通量测序文库简易构建的载体制作方法,以解决上述背景技术中提出的问题。
为了解决上述技术问题,本发明提供如下技术方案:增强子筛选高通量测序文库简易构建的载体制作方法,包括以下步骤:
步骤一,构建含有SpeI单酶切位点和EcoRI酶切位点的质粒1;
步骤二,使用多重PCR扩增待测增强子群;
步骤三,链接高通量测序接头序列1和2,使用引物1和引物2进行PCR扩增,构建含有待测增强子的DNA文库(DNA文库1);
步骤四,使用引物3和引物4,PCR扩增DNA文库1,得到DNA文库2;
步骤五,使用SpeI和EcoRI双酶切质粒1和DNA文库2,酶联两者的双酶切产物,构建成结构含有多个待测增强子的质粒群2;
步骤六,使用引物1和引物2,PCR扩增质粒群2,得到DNA测序文库(DNA文库3),满足Illumina平台高通量测序仪的上样要求;
步骤七,用质粒群2瞬时转染细胞,抽提RNA;使用引物2进行反转录,得到cDNA;使用引物1和引物2进行PCR,得到RNA测序文库(DNA文库4),满足Illumina高通量测序的上样要求。
与现有技术相比,本发明所达到的有益效果是:本发明通过多重PCR方式,扩增待测增强子群,可以将扩增样本中的罕见突变保存下来。本发明的设计使得对其所含待测增强子进行高通量测序文库构建的步骤由常规步骤(包含超声打断、末端修复、加接头、PCR)变成了PCR一步。本发明的设计使得对其所含待测增强子的转录本RNA进行高通量测序文库构建的步骤由常规步骤(包含oligo dT反转录,末端修复、加接头、PCR)变成了引物2反转录和PCR两步。此外,使用引物2反转录,避免了oligo dT反转录过程中将大量无关的mRNA转录,降低了后续测序成本和数据分析难度。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1是本发明的质粒1结构示意图。
图2是本发明的质粒群2的结构示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅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。
请参阅图1-2,本发明提供一种技术方案:增强子筛选高通量测序文库简易构建的载体制作方法,包括以下步骤:
步骤一,构建含有SpeI单酶切位点和EcoRI酶切位点质粒1;
步骤二,使用多重PCR扩增待测增强子群;
步骤三,链接高通量测序接头序列1和2,使用引物1和引物2进行PCR扩增,构建含有待测增强子的DNA文库(DNA文库1);
步骤四,使用引物3和引物4,PCR扩增DNA文库1,得到DNA文库2;
步骤五,使用SpeI和EcoRI双酶切质粒1和DNA文库1,酶联两者的双酶切产物,构建成结构含有多个待测增强子的质粒群2;
步骤六,使用引物1和引物2,PCR扩增质粒群2,得到DNA测序文库(DNA文库3),满足Illumina平台高通量测序仪的上样要求;
步骤七,用质粒群2瞬时转染细胞,抽提RNA;使用引物2进行反转录,得到cDNA;使用引物1和引物2进行PCR,得到RNA测序文库(文库4),满足Illumina高通量测序的上样要求。
在质粒群2中:
序列1:5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
序列2:5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXATCTCGTATGCCGTCTTCTGCTTG
(其中XXXXXXX是可变化的序列)
引物1:5’-AATGATACGGCGACCACCGA
引物2:5’-CAAGCAGAAGACGGCATACGA
引物3:5’-AACTACTAGTAATGATACGGCGACCACCGA
引物4:5’-TATGAGAATTCCAAGCAGAAGACGGCATACGA
本发明通过多重PCR方式,扩增待测增强子群,可以将扩增样本中的罕见突变保存下来。
本发明中质粒群2的设计使得对其所含待测增强子进行高通量测序文库构建的步骤由常规步骤(包含超声打断、末端修复、加接头、PCR)变成了PCR一步。
本发明中质粒群2的设计使得对其所含待测增强子的转录本RNA进行高通量测序文库构建的步骤由常规步骤(包含oligo dT反转录,末端修复、加接头、PCR)变成了引物2反转录和PCR两步。此外,使用引物2反转录,避免了oligo dT反转录过程中将大量无关的mRNA转录,降低了后续测序成本和数据分析难度。
以下为质粒群2中含有SV40增强子序列的质粒序列:
GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCTGCGATCTGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAATTGGACTAGTCGATGGAGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGCGAATTCCGAGTCGGGGCGGCCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCCCAAGCTACCATGATAAGTAAGTAATATTAAGGTACGGGAGGTACTTGGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGTACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGATA
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.增强子筛选高通量测序文库简易构建的载体制作方法,其特征在于,包括以下步骤:
步骤一,构建含有SpeI单酶切位点和EcoRI酶切位点质粒1;
步骤二,使用多重PCR扩增待测增强子群;
步骤三,链接高通量测序接头序列1和2,使用引物1和引物2进行PCR扩增,构建含有待测增强子的DNA文库(DNA文库1);
步骤四,使用引物3和引物4,PCR扩增DNA文库1,得到DNA文库2;
步骤五,使用SpeI和EcoRI双酶切质粒1和DNA文库2,酶联两者的双酶切产物,构建成结构含有多个待测增强子的质粒群2;
步骤六,使用引物1和引物2,PCR扩增质粒群2,得到DNA测序文库(DNA文库3),满足Illumina平台高通量测序仪的上样要求;
步骤七,用质粒群2瞬时转染细胞,抽提RNA;使用引物2进行反转录,得到cDNA;使用引物1和引物2进行PCR,得到RNA测序文库(DNA文库4),满足Illumina高通量测序的上样要求。
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Cited By (3)
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CN110257430A (zh) * | 2019-06-19 | 2019-09-20 | 中国农业科学院农业基因组研究所 | 一种活性增强子筛选载体及其构建方法 |
CN110885845A (zh) * | 2019-06-19 | 2020-03-17 | 中国农业科学院农业基因组研究所 | 一种基于随机序列利用载体筛选活性增强子的方法 |
CN116286991A (zh) * | 2023-02-10 | 2023-06-23 | 中国农业科学院农业基因组研究所 | 全基因组增强子筛选系统、筛选方法及应用 |
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