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    Genetic analysis using peptide tagged in-vitro synthesized proteins
    3.
    发明授权
    Genetic analysis using peptide tagged in-vitro synthesized proteins 有权
    使用肽标记的体外合成蛋白质的遗传分析

    公开(公告)号：US06329180B1

    公开(公告)日：2001-12-11

    申请号：US09266462

    申请日：1999-03-11

    申请人： Alex M. Garvin

    发明人： Alex M. Garvin

    IPC分类号： C12Q168

    CPC分类号： C12Q1/6827 ,  C12Q2565/627 ,  C12Q2561/127

    摘要： A method and kit are disclosed useful for detecting protein altering mutations in genes. The coding sequence of the gene is PCR amplified with a 5′ primer that contains at its 5′ end a polymerase binding site, a translation initiation site, and an in frame sequence coding for a peptide tag, followed by the in frame 5′ end of the test sequence. The coding sequence for the peptide tag can also be incorporated into the 3′ primer used for PCR. After PCR amplification of the test sequence, the PCR product is used as a template to make mRNA in an in-vitro transcription reaction using an RNA polymerase that recognizes the polymerase binding site incorporated into the 5′ PCR primer. The mRNA is then used as a template to make protein in an in-vitro translation reaction. The protein encoded by the test sequence has at its amino or carboxy terminus a peptide tag that can be used to purify the protein for further analysis. For example, the mass of the purified protein can be determined by mass spectrometry in order to detect protein-altering mutations. Examples are disclosed using matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry instruments to analyze proteins of interest.

    摘要翻译： 公开了用于检测基因中的蛋白质改变突变的方法和试剂盒。 基因的编码序列用5'引物PCR扩增，其5'末端包含聚合酶结合位点，翻译起始位点和编码肽标签的帧序列，随后在框架5'末端 的测试序列。 肽标签的编码序列也可以并入用于PCR的3'引物中。 在PCR扩增测试序列之后，使用PCR产物作为模板，使用识别融合到5'PCR引物中的聚合酶结合位点的RNA聚合酶使mRNA进行体外转录反应。 然后将mRNA用作模板以在体外翻译反应中制备蛋白质。 由测试序列编码的蛋白质在其氨基或羧基末端具有可用于纯化蛋白质用于进一步分析的肽标签。 例如，可以通过质谱法测定纯化的蛋白质的质量，以便检测蛋白质改变的突变。 公开了使用基质辅助激光解吸电离（MALDI）飞行时间（TOF）质谱仪器来分析目的蛋白质的实例。