分子标记技术及其应用ISSR.ppt

1、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（

2、microsatellite-primed PCR, MP-PCR）技术。,10.1 Introduction,ISSR标记技术由加拿大蒙特利尔大学的Zietkiewicz等于1994年提出。,用锚定的微卫星DNA为引物，即在SSR序列的3端或5端加上24个随机核苷酸，在PCR反应中，锚定引物可以引起特定位点退火，导致与锚定引物互补的间隔不太大的重复序列间DNA片段进行PCR扩增。所扩增的inter SSR区域的多个条带通过聚丙烯酰胺凝胶电泳或者琼脂糖凝胶电泳得以分辨，扩增带多为显性表现。,10.2 Principle of ISSR,锚定ISSR-PCR示意图,用重复序列(CA)n作单引物，

3、在引物的5 端或3锚定1至数个碱基。,ISSR遗传多态性高，重复性好。（1724bp） 符合孟德尔遗传规律。 无需知道任何靶标序列的SSR背景信息。 DNA用量少,10.3 Characteristic,Advantages：,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,disadvantages：,DNA extraction and purification,10.4 Procedure,Primer design,ISSR引物常为5端或3 加猫（24个碱基）的二核苷酸、三核苷酸、四核苷酸序列，重复次数常为48次。,PCR amplification,6 PAG

4、E,Detect of PCR product and statistical analysis,扩增步骤与RAPD相似，但不同引物、不同材料的扩增条件有异。,Primer 0.20.8mol/L Template DNA 550ng Taq DNA polymerase 0.41U,需要做预备实验优化PCR条件，以获得清晰、可重复，易统计的条带。,25l,0.52.0% Agarose gel electrophoresis,EB staining,Silver staining,yes,1,No,0,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

5、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：,聚合反应催化剂配伍,Note：(NH4)2S2O8，过硫酸胺 TEMED：N，N，N，N;四甲基乙二胺 DMAPN：二甲基胺基丙晴 用过硫酸铵引发的反应称化学聚合反应；用核黄素引发，需要强光照射反应液， 称光聚合反应。,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

6、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。,Pouring Acrylamide Gels,Preparation of glass plates,Wash the glass plates thoroughly in water and soap with a sponge. Rinse with dH2O, and dry with paper towels. Rub the short plate with 95% ethanol. Disper

7、se 1 ml bind-silane on the glass plate and let it dry for 5 min. Rub the plate with a wet (ddH2O) paper towel. Rinse the plate with ddH2O and rub it dry with a paper towel. Rub the plate with 95% ethanol Rub the long glass plate with 95% ethanol Disperse 2 ml repel-silane thoroughly on the glass pla

8、te. Rub the plate with a few drops ddH2O.,Caution unpolymerized acrylamide is toxic; all steps should be done wearing gloves.,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 botto

9、m 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 cover

10、ed 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

11、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 re

12、move the dried, polymerized gel. Carefully remove the comb, and clean the comb and well with 1x TBE.,Pouring the gel,Final conc.For 100 ml 250 ml 500 ml solid Urea 10 mM 36g 90g 180g 40% Acrylamide solution 4 % 10 ml 25 ml50 ml 10 x TBE 1x 10 ml 25 ml50 ml ddH2O 51 ml 125 ml250 ml,Solutions and Reag

13、ents,Acrylamide/bisacrylamide stock,10% APS solution,Dissolve 5 g ammonium persulfate (APS) in 50 ml ddH2O. Aliquot 600 ml into 1.6 ml microfuge tubes and store at -20.,Final conc.For 100 ml 250 ml 500 ml Acrylamide 38 % 38 g95 g 190 g Bis-crylamide 2 % 2 g 5 g 10 g ddH2O 50 ml 125 ml 250 ml Dissolv

14、e by stirring at 37, adjust volume to final with ddH2O.,40% Acrylamide solution (19:1 acrylamide/bis-acrylamide),Mount the gel with the short glass plate inwards in the electrophoresis apparatus. Make sure that the buffer valve is closed. Pour 500 ml 1x TBE into the upper chamber and rinse between t

15、he plates with a syringe containing some buffer. If no buffer leaks from the upper chamber can be found, 500 ml 1x TBE can be poured into the lower chamber. Connect the apparatus to a power supply and turn on the power with the negative (black) lead attached to the top chamber and the positive (red)

16、 to the bottom. Set the supply at 2500 V, 80 mA and 80W. Let the gel prerun for at least 30 min,Running Acrylamide Gels,Installation of plates,Add 10 ml formamide loading buffer to each probe (8 ml volume from the PCR reaction). Denature the probes in a thermocycler: 2 min. at 94 for SSR samples. Im

17、mediately store the probes on ice. Disconnect the power supply and rinse the cavity between the glass plates once more. Place the comb with the teeth towards the gel and push the teeth slightly into the gel taking care not to tear the gel. Load 5 ml of each sample between the teeth with a capillary

18、pipette. Load DNA size standards on the flanking sides or as separators between different kinds of probes.,Preparing and loading first set of probes,Reconnect the power supply, and run the gel (2500 V, 80 mA, and 80W). After first samples have run into the gel for 15 min, other reactions from the sa

19、me primer set can be loaded on the same gel (up to 4 consecutive loadings can be performed). Be sure to turn off the power supply between loadings. If 72 well combs are used with 4 consecutive loadings, ie. multiplexed loadings, a mapping population of 240 individuals can be run on a single gel, max

20、imizing data per gel while minimizing the number of gels required for the analysis of a whole population. When the first color marker (bromophenol blue) from the first loading runs out of the gel, the gel run is complete (about 1 h. 15 min. elapsed runtime at 2500 V). Note: the run time can vary wit

21、h the fragment size to be detected. When the gel run is complete, turn off the power, and disconnect the leads. Drain the buffer from the upper chamber into a large beaker via the valve. Demount the gel.,Running the gel and Multiplex loading,Remove the comb and side spacers of the gel. Separate the

22、glass plates with a metal spatula. The gel will bind to the short plate and be released from the long plate. Place the short plate with the gel in a metal tray containing 2 L fix/stop solution and agitate for at least 30 min (the blue color of the loading buffer should be washed out). Rinse the gel

23、in 2 L water for 2 x 4 min and 1 x 2 min. Transfer the gel to the staining solution and agitate for 30 min. The staining solution can be used three times. The second and third time the gel must stain for 40 and 50 min, respectively. Prepare a tray with 4 L of water and another with developing soluti

24、on (add formaldehyde and sodium thiosulphate to the developing solution just prior to use). Lif the gel up and down three times into the water containing tray and immediately transfer the gel to the developing solution. Agitate the gel until the bands develop (2-3 min). Terminate the developing reaction and fix the gel by adding the 2 L of fix/stop solution used previously. Agitate until the solution stops fizzing (2-3 min). Rinse the gel in distilled water 2 x 5 min. Air dry. Pour the silver stain into its contai