一种新型DNA标记技术——限制性位点扩增多态性(RSAP)的建立与优化

Development and refinement of a new marker technique Restriction site amplified polymorphism(RSAP)

了避开基于限制性酶切位点分子标记技术的DNA酶切环节，简化其复杂的程序，试验以辣椒为材料，建立了一种新型DNA标记技术——限制性位点扩增多态性（RSAP），并对其反应体系进行了优化，对RSAP适用性、重复性进行了检验。结果显示，RSAP技术的引物为2条长度均为18bp的引物，引物的3’端为4～6个碱基的限制性酶切位点序列，接着是12～14个碱基的随机序列，2条引物的限制性位点和随机序列不同；PCR扩增的前5个循环采用35C的退火温度，随后的35个循环采用48℃的退火温度；在25μL反应体系中，模板DNA用量为20ng，Mg^2＋浓度为2．5mmol／L，dNTPs浓度为0．2mmol／L，TaqDNA聚合酶用量为1．5U，2条引物浓度均为600nmol／L;RSAP技术重复性好，适用性广泛，是一种操作简便、产率中等、稳定可靠的DNA标记技术。

To avoid DNA digestion in some maker techniques based on restriction sites and simplify complex procedure of these techniques,a new marker technique called restriction site amplified polymorphism （RSAP） was developed and refined with pepper DNA as trial material. The reproducibility and broad application were tested as well. The results showed that RSAP used two primers of 18 nucleotides in which starting at the 3＇ end of each primer were restriction site sequences （4-6 bases）,followed 12-14 bases of arbitary sequence,the differences between two primers were restriction sites and filler sequence;PCR amplification was run for the first 5 cycles with an annealing temperature of 35 ℃ ,followed by 35 cycles with an annealing temperature of 48 ℃;the PCR reaction of 25μL included 20 ng DNA templates,2.5 mmol/L of Mg^2＋,0. 2 mmol/L of dNTP,1.5 U of Taq DNA polymerase,600 nmol/L of each primers. RSAP is a new maker technique with simplicity,moderate throughput ratio and reliability. RSAP is of good reprodicibity and broad application.