| 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.
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