运用一步化体外转录-翻译系统筛查基因突变的新技术

Gene Mutation Screening by Using Coupled In Vitro Transcription and Translation System

报道了一种筛查基因突变的新技术──PCR结合一步化体外转录－翻译系统。该技术包括：（1）PCR扩增靶DNA片段，并使DNA产物的一端或两端带上T7T1序列（T1：翻译起始信号，CCACCATG）；（2）以该PCR产物为模板，利用一步化体外转录－翻译系统（CoupledInVitroTranscription－TranslationSystem）合成相应肽链；（3）用SDS－PAGE及高分辨pH梯度PAGE（聚丙烯酰胺凝胶电泳）分离分析多肽产物，通过异常电泳图谱而发现基因突变。本文以已知突变的乙型血友病（HemophiaB，即HB）患者DNA及正常人DNA为标本，建立了上述技术方法。4个HB患者FIX基因外显子H内的无突变，在SDS-PAGE的射自显影图谱上全部显示出变短了的多肽区带。11个在外显子E中有错义突变的标本中，有10例可用pH梯度PAGE检出。说明运用PCR及体外一步化转录－翻译系统，并结合SDS－PAGE或PH梯度PAGE，是一种能快速、灵敏地筛查基因突变的技术，值得进一步研究推广。

A new method for screening gene mutations is reported in this poper. It includes the following steps:(1) The target DNA fragment with T7T1 sequence in its 5' end is amplified (T7T1:GGATCCTAATACGACTCTATAGGGAGACCACCATG); (2) The peptide is synthesized and labled from the PCR product in a Coupled In Vitro Transcription-Translation System; (3) The produced peptides are analyzed by using SDS-PAGE and pH Gradient PAGE. Four peptide products from 4 HB patients with nonsense mutation in Exon H showed truncated protein bands with speeded migration in the autoradiography of the SDS-PAGE. Ten out of 11 HB patients with different missense mutations showed abnormal patterns in the autoradiography of a pH 4 -7 gradient gel focusing electrophoresis. Conclution: The PCR and the Coupled Transcription-Translation System combined SDS-PAGE is a good method for proteins truncation tast. The PCR and the Coupled Transcription-Translation System combined pH Gradient PAGE is an efficient method for screening the abnormal protein products from DNA fregments with mutations.