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An easy pcr-based genome-walking method for getting the unknown 5’flanking region of a Scenedesmus sp.

Journal of Coastal Life Medicine 2015年第5期 348-351页

作者： Ahmed Elsayed Gomma Jin Man Kim Seung Hwan Yang Gyuhwa Chung Department of Biotechnology Chonnam National UniversityYeosuChonnam 550-749Korea Center for Nutraceutical and Pharmaceutical Materials Myongji UniversityCheoin-guYonginGyeonggi-Do 449-728Korea

Objective:To develop the current single primer pcr-based genome-walking method with Scenedesmus ***:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer pcr-based genome-walking method were *** was between the related species of microalga and Scenedesmus *** 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas *** the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas ***:Obtaining a small conserved sequence for any gene family is something that can be achieved quite ***,identifying the whole gene is often *** investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer pcr-based genome-walking *** performed two consecutive pcr reactions;band extraction and the pcr product were *** got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each *** also tested two different DNA polymerases Ex Taq and TLA ***:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.

关键词： Single primer pcr pcr-based genome-walking rbcS rbcL 18S rDNA 5’flanking region Ex Taq TLA polymerase

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