中国医科大学《生物化学》课件-第17章 分子生物学基本技术定稿.pptVIP

* Serratia marcescens 粘质沙雷菌(S. marcescens) Mycobacterium phlei 草分枝杆菌(M. phlei) * DNA hybridization. Two DNA samples to be compared are completely denatured by heating. When the two solutions are mixed and slowly cooled, DNA strands of each sample associate with their normal complementary partner and anneal to form duplexes. If the two DNAs have significant sequence similarity, they also tend to form partial duplexes or hybrids with each other: the greater the sequence similarity between the two DNAs, the greater the number of hybrids formed. Hybrid formation can be measured in several ways. One of the DNAs is usually labeled with a radioactive isotope to simplify the measurements. * 马铃薯基因组DNA经EcoRI消化后的图谱 * 【A】Enlarged image of a DNA microarray. Each glowing spot in this microarray contains DNA from one of the 6,200 genes of the yeast (S. cerevisiae) genome, with every gene represented in the array. The microarray has been probed with fluorescently labeled nucleic acid derived from the mRNAs obtained (1) when the cells were growing normally in culture and (2) five hours after the cells began to form spores. The green spots represent genes expressed at higher levels during normal growth; the red spots, genes expressed at higher levels during sporulation. The yellow spots represent genes that do not change their levels of expression during sporulation. This image is enlarged; the microarray actually measures only 1.8 × 1.8 cm. 一、PCR基本原理 ◆ 是在热稳定DNA聚合酶催化下的体外DNA合成的循环反应。 ◆ PCR反应体系：DNA模板、热稳定性DNA聚合酶、引物、四种dNTP和Mg2＋。 ◆ PCR反应三个基本过程： (1) 变性：利用高温(~95℃)使双链解开。 (2) 退火：在低温(25~65℃)下使引物与模板配对。 (3) 延伸：在~72℃利用热稳定DNA聚合酶催化DNA合成。 模板 DNA 引物 dNTP Taq DNA Pol 变性 退火 延伸 PCR循环 ◆ PCR反应三个基本过程可以不断循环，从而使体系内DNA的量呈指数增长。 变性 退火 延伸 变性 退火 延伸 循 环 1 循 环 2 经过多轮PCR 二、PCR反应试剂 1. 模板 (1) 模板DNA用量少，一般使用102~105拷贝 (2) 有一定的纯度要求 (3) 对模板的序列有一定了解 2. 引物 ◆ 引物为一段寡核苷酸序列。 ◆ 每条引物的典型浓度约0.1~0.5μmol/L ◆ 引物设计一般原则： (1) 为待扩增序列两端的已知序列 (2) 长度一般为18-30个寡核苷酸 (3) G+C含量合理：40~60%，Tm＝4(G+C)＋2(A+T)