【摘要】：目前,PCR引物設(shè)計(jì)主要依賴于軟件對(duì)引物熔點(diǎn)的模擬計(jì)算,而PCR退火條件的優(yōu)化需進(jìn)行不同條件下的擴(kuò)增實(shí)驗(yàn)。為開(kāi)發(fā)一種可高效、精確評(píng)價(jià)引物和確定退火條件的方法,本研究采用高分辨率熔解曲線(high resolution melting,HRM)測(cè)定技術(shù)直接分析短鏈DNA的熔點(diǎn),用于引物優(yōu)劣性的評(píng)價(jià),并為退火條件的優(yōu)化提供參考。本文用HRM法直接測(cè)定了非完全互補(bǔ)的雙鏈DNA以及DNA發(fā)卡結(jié)構(gòu)的熔點(diǎn),結(jié)果顯示:(1)與完全互補(bǔ)的雙鏈DNA相比,較為穩(wěn)定的單堿基錯(cuò)配AsIG、GsIG和TsIG的熔點(diǎn)只降低2℃~3℃,部分雙堿基錯(cuò)配的熔點(diǎn)只降低4℃~6℃,單堿基突出熔點(diǎn)只降低4℃~5℃。因此,如果采用的退火溫度不當(dāng),部分錯(cuò)配的非目的模板可能會(huì)被擴(kuò)增。(2)即使發(fā)卡結(jié)構(gòu)的莖干區(qū)只有6 bp,當(dāng)其環(huán)區(qū)堿基少于10 nt時(shí),其熔點(diǎn)也可達(dá)到60℃以上。此外,環(huán)區(qū)的長(zhǎng)度對(duì)發(fā)卡熔點(diǎn)也有較大影響。根據(jù)本研究結(jié)果發(fā)現(xiàn),引物設(shè)計(jì)時(shí)應(yīng)盡量避免模板引物結(jié)合區(qū)同其鄰近的30 nt堿基有6 bp以上的互補(bǔ)部分。綜上所述,本研究證明HRM熔點(diǎn)法是一種高效評(píng)價(jià)引物及確定退火溫度的方法。
[Abstract]:At present, the design of PCR primer mainly depends on the simulation calculation of the melting point of the primer by software, but the optimization of PCR annealing conditions needs to carry out amplification experiments under different conditions. In order to develop an efficient and accurate method for evaluating primers and determining annealing conditions, the melting point of short-chain DNA was directly analyzed by high-resolution melting curve (high resolution melting,HRM, which was used to evaluate the advantages and disadvantages of primers. It also provides a reference for the optimization of annealing conditions. In this paper, the melting point of incomplete complementary double-stranded DNA and DNA card issuing structure has been directly determined by HRM method. The results show that: (1) compared with fully complementary double-stranded DNA, the melting point of stable single-base mismatch AsIG,GsIG and TsIG is only 2 鈩，

本文編號(hào)：2356895