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1. 发明授权

US5962272A Methods and compositions for full-length cDNA Cloning using a template-switching oligonucleotide 失效
标题翻译：使用模板切换寡核苷酸的全长cDNA克隆的方法和组合物
公开(公告)号：US5962272A
公开(公告)日：1999-10-05
申请号：US778494
申请日：1997-01-03
申请人： Alex Chenchik , York Zhu , Luda Diatchenko , Paul Siebert
发明人： Alex Chenchik , York Zhu , Luda Diatchenko , Paul Siebert
IPC分类号： C12N15/09 , C12N15/10 , C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12Q1/6855 , C12N15/1096
摘要： The present invention pertains to methods for the synthesis and cloning of full-length cDNA, or cDNA fragments, that correspond to the complete sequence of 5'-ends of mRNA molecules. The method of the present invention comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5'-end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3'-end of the ss cDNA that is complementary to the template switching oligonucleotide. The resulting full-length ss cDNA includes the complete 5'-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA.The subject invention also pertains to the template switching oligonucleotides that can be used according to the subject method. Kits containing the template switching oligonucleotide are also included within the scope of the present invention.
摘要翻译：本发明涉及合成和克隆与mRNA分子的5'末端的完整序列相对应的全长cDNA或cDNA片段的方法。本发明的方法包括使RNA与可以与RNA退火的cDNA合成引物，其具有逆转录酶活性的合适的酶，以及模板切换寡核苷酸，其条件是足以允许引物的模板依赖性延伸以产生 mRNA-cDNA杂交。模板切换寡核苷酸与RNA分子的5'末端的CAP位点杂交，作为与模板切换寡核苷酸互补的ssDNA的3'-末端的CAP依赖性延伸的短扩展模板。所得的全长ss cDNA包括RNA分子的完整5'-末端以及与模板切换寡核苷酸互补的序列，其随后可作为cDNA随后扩增中的通用启动位点。本发明还涉及根据本发明方法可以使用的模板切换寡核苷酸。含有模板切换寡核苷酸的试剂盒也包括在本发明的范围内。

2. 发明授权

US5962271A Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end 失效
标题翻译：用于产生在3'末端具有任意核苷酸序列的全长cDNA的方法和组合物
公开(公告)号：US5962271A
公开(公告)日：1999-10-05
申请号：US582562
申请日：1996-01-03
申请人： Alex Chenchik , York Zhu , Luda Diatchenko , Paul Siebert
发明人： Alex Chenchik , York Zhu , Luda Diatchenko , Paul Siebert
IPC分类号： C12N15/09 , C12N15/10 , C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12Q1/6855 , C12N15/1096
摘要： Described are compositions and methods which allow for the efficient addition of a defined sequence at the 3'-end of a full-length cDNA in the course of first-strand cDNA synthesis from an mRNA template. A cDNA synthesis primer that is capable of annealing to mRNA is used to prime the first strand synthesis reaction. An oligonucleotide that is linked to the 5'-end of the mRNA serves as a short, extended template such that when the reverse transcriptase enzyme reaches the 5'-end of the mRNA, the enzyme switches templates and proceeds to transcribe through the end of the linked oligonucleotide. As a result, the single-stranded cDNA product which corresponds to the full-length mRNA, will have at the 3'-end a defined sequence which is complementary to the linked oligonucleotide. A conservative element in the oligonucleotide sequence responsible for this reaction can include 3 to 5 guanylic acid residues at the 3'-end of the oligonucleotide. The subject invention provides for the increased synthesis of full-length cDNA from mRNA templates. The full-length cDNA prepared according to the present invention can then be amplified using PCR or cloned using standard procedures.
摘要翻译：描述了允许在mRNA模板的第一链cDNA合成过程中在全长cDNA的3'-末端有效添加限定序列的组合物和方法。使用能够对mRNA退火的cDNA合成引物用于引发第一链合成反应。与mRNA的5'-末端连接的寡核苷酸用作短的扩展模板，使得当逆转录酶达到mRNA的5'末端时，酶切换模板并继续转录到连接的寡核苷酸。结果，对应于全长mRNA的单链cDNA产物将在3'末端具有与连接的寡核苷酸互补的确定的序列。负责该反应的寡核苷酸序列中的保守元件可以在寡核苷酸的3'-末端包括3至5个鸟苷酸残基。本发明提供了从mRNA模板增加的全长cDNA的合成。然后可以使用PCR扩增根据本发明制备的全长cDNA或使用标准程序克隆。

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