Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific singlestranded DNA and improved real-time detection
Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific singlestranded DNA and improved real-time detection
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DOI：
10.1073/pnas.0501946102
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发表时间：
2005-06-14
影响因子：
11.1
通讯作者：
Wangh, LJ
中科院分区：
文献类型：
--
作者：
Pierce, KE;Sanchez, JA;Wangh, LJ
传统的不对称PCR使用不同浓度的常规PCR引物来产生单链DNA。然而，这种方法难以优化，通常效率低下，并且倾向于促进非特异性扩增。另一种方法，指数后线性（LATE）-PCR，解决了这些问题，通过使用引物对故意设计用于在不相等的浓度。本报告系统地研究了影响LATE-PCR扩增的指数和线性阶段的引物设计参数。特别地，我们研究了在LATE-PCR的指数期期间，相对于过量引物（T-m（X））改变限制引物（T-m（L））的浓度调节的解链温度（T-m）如何影响扩增效率和特异性。当T-m（L）- T-m（X）>= 5 ℃时，观察到最高的反应效率和特异性。我们还研究了相对于双链扩增子的较高T（T-m（A））改变T-m（X）如何影响线性扩增的速率和程度。具有更接近T-m（A）的T-m（X）的过量引物产生更高的线性扩增速率和来自杂交探针的更强的信号。这些设计标准最大限度地提高了特异性单链DNA产物的产量，并使LATE-PCR更稳健，更容易实施。通过使用扩增囊性纤维化跨膜调节因子（CFTR）基因内的序列的引物对来验证结论，所述基因的突变负责囊性纤维化。
Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (T-m) of the limiting primer (T-m(L)) relative to that of the excess primer (T-m(X)) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when T-m(L) - T-m(X) >= 5 degrees C. We also investigated how altering T-m(X) relative to the higher T of the double-stranded amplicon (T-m(A)) affects the rate and extent of linear amplification. Excess primers with T-m(X) closer to T-m(A) yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.