分子标记技术及其应用 12Chapter12 STS.ppt

1、Chapter12 STS ( Sequence Tagged Site),由Olson于1989年开发成功。 STS（Sequence-Tagged Site）序列标签位点: 是基因组上定位明确、作为界标并能通过PCR扩增被唯一操作的短的、单拷贝DNA 序列，用于产生作图位点，即测定一系列STS 的次序即可作出基因组区域的图谱。 STS include: Simple Sequence Repeat Polymorphisms (SSR or SSRP), Anchored Simple Sequence Repeats (ASSR), Sequence-characterized ampl

2、ified regions (SCAR), Cleaved Amplified Polymorphism Sequences (CAPS), Single Primer Amplification Reaction ( SPAR) and so on.,12.1 Introduction,Cleaved Amplified Polymorphism Sequences酶切扩增多态性序列 CAPS标记（Konieczny and Ausubel 1993）是特异引物PCR与限制性酶切相结合而产生的一种DNA标记，它实际上是一些特异引物PCR标记（如SCAR和STS）的一种延伸。当SCAR或STS

3、的特异扩增产物的电泳谱带不表现多态性时，一种补救办法就是用限制性内切酶对扩增产物进行酶切，然后再通过琼脂糖或聚丙烯酰胺凝胶电泳检测其多态性。用这种方法检测到的DNA多态性就称为CAPS标记。它揭示的是特异PCR产物DNA序列内限制性酶切位点变异的信息，也表现为限制性片段长度的多态性。,CAPS,Single Primer Amplification Reaction单引物扩增反应 Gupta et al. , 1994. This technique is based on PCR (polymerase chain reaction) amplification of DNA markers

4、, using single primers of simple sequence repeats (SSR) or microsatellites. SPAR技术与RAPD技术相似的是也只用一个引物，但SPAR分析中所用的引物不是随机的，而是在SSR的基础上设计的，例如可能的序列是(TA)10或(CGA)6。扩增的是SSR之间的DNA序列。又称为MP-PCR (microsatellite-primed PCR) 。,SPAR,Sequence tagged site (STS): Short (200 to 500 base pairs) DNA sequence that has a s

5、ingle occurrence in the genome and whose location and base sequence are known. Detectable by polymerase chain reaction, STSs are useful for localizing and orienting the mapping and sequence data reported from many different laboratories and serve as landmarks on the developing physical map of the ge

6、nome. STS是指基因组中长度为200-500bp，且核苷酸顺序已知的单拷贝序列，通过PCR可将其专一扩增出来。,12.2 Concept and principle of STS,Concept,基本原理: 依据 单拷贝的RFLP探针、微卫星序列、Alu因子等两端序列，设计合适的引物，进行PCR扩增，电泳显示扩增产物多态性。有时扩增产物还需要特定的限制性内切酶酶解后才能表现出多态性。目前用于STS引物设计的主要是RFLP探针。,Principle,标记来源广，数量多； 共显性遗传； STS在基因组中往往只出现一次，从而能够界定基因组的特异位点; 技术简便，检测方便； STS标记采用常规P

7、CR所用的引物长度，结果稳定可靠。 与SSR标记一样，开发依赖于序列分析及引物合成，成本较高； 多态性常常低于相应的RFLP等标记，这是因为STS仅仅检测该引物所分布区域的片段差异或酶切位置差异，而RFLP标记的多态性往往可能是探针以外区域的差异，这一部分差异无法转化成STS标记的多态性。,12.3 Characteristic,DNA extraction and purification,12.4 Procedure,PCP-product Digestion,Steps-1,Thaw the components of the following mastermix. Keep the

8、buffer and the enzyme on ice. Label microfuge tubes corresponding to the samples used for STS analysis. Transfer 10.0 l of the PCR reactions product into the labeled tubes. Prepare the mastermix (on ice):,Mix mastermix and centrifuge down. Pipette 10.0 ml of the mastermix into each microfuge tube. Mix components, and centrifuge down. Place the tubes at the required temperature (for most restriction endonucleases this is