分子标记技术及其应用ISSR.pptVIP

Chapter10 ISSR (Inter-Simple Sequence Repeat) 在SSR标记技术中，采用PCR反应必须知道扩增DNA片段两侧序列，在大多数情况下，某些序列本身或其旁测序列并不清楚，而且由于SSR两侧引物具有物种特异性，引物设计费时耗力，要检测多个基因座是不现实的。这就限制了PCR技术的应用，“锚定PCR（anchored PCR）”可克服上述障碍。锚定简单重复序列（anchored simple sequence repeat，ASSR），也称作简单重复序列间区（inter-simple sequence repeat，ISSR）标记技术或者以微卫星为引物的PCR（microsatellite-primed PCR, MP-PCR）技术。 PCR扩增反应的最适条件需要一定时间摸索。 ISSR标记大多是显性标记 PAGE 聚丙烯酰胺凝胶电泳是以聚丙烯酰胺凝胶作为支持介质的电泳方法。在这种支持介质上可根据被分离物质分子大小和分子电荷多少来分离。 在聚丙烯酰胺凝胶形成的反应过程中，需要有催化剂参加，催化剂包括引发剂和另速剂两部分。引发剂在凝胶形成中提供始自由基，通过自由基的传递，使丙烯酰胺 成为自由基，发动聚合反应，加速剂则可加快引发剂放自由基的速度。常用的引发剂 和加速剂的配伍如下表： 聚丙烯酰胺聚合反应可受下列因素影响 1、大气中氧能淬灭自由基，使聚合反应终止，所以在聚合过程中要使反应液与空气 隔绝。 2、某些材料如有机玻璃，能抑制聚合反应。 3、某些化学药物可以减慢反应速度，如赤血盐。 4、温度高聚合快，温度低聚合慢。 以上几点在制备凝胶时必须加以注意。 Precook the gelstock if necessary. Place the side spacers at the edge of the long plate and place the short glass plate above. Place one spring clip on each side at the bottom of the gelform. Mix the following components in a beaker 60 ml 4 % gelstock 60 ml TEMED 500 ml APS Cast the gel immediately after the addition of the APS, by injecting the gel solution with a syringe from the glass beaker between the glass plates at the top (where the long glass plate is not covered by the short glass plate). A slight incline of the gelform facilitates faster gel casting. Make sure not to introduce any bubbles! When the gelform is full, insert the flat edge of the combe at the top between the plates, about 5 mm below the edge of the short glass plates. Place spring clips on each side of the gel plates, one at the middle and one near the top. To insure tight contact between the plates and the comb, place two spring clips over the top of the glass plates. Let the gel polymerize for about 1 hour. Remove the spring clips over the comb, and rinse this area with 1x TBE to remove the dried, polymerized gel. Carefully remove the comb, and clean the comb and well with 1x TBE. Final conc. For 100 ml 250 ml