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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis
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Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

机译：指数线性（LATE）-PCR：一种不对称PCR的先进方法及其在定量实时分析中的应用

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摘要

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)-PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80-250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic f ibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.
机译：常规的不对称PCR效率低下并且难以优化，因为限制一种引物的浓度将其熔化温度降低到低于反应退火温度。指数线性（LATE）-PCR描述了引物设计的新范例，无论引物比例如何，该方法都使测定与对称PCR一样高效。 LATE-PCR生成单链产物，其在指数期以外的许多循环中具有可预测的动力学。 LATE-PCR还引入了新的探针设计标准，该标准可将杂交探针的检测与引物退火和延伸脱钩，提高探针的可靠性，改善等位基因的分辨力，并使信号强度相对于对称PCR增加80-250％。 PCR的这些改进对于小样本中目标数量的实时定量分析特别有用。 LATE-PCR适用于临床诊断，生物防御，法医和DNA测序等领域的高通量应用。我们展示了通过扩增单个杂合性细胞的囊性纤维化CFΔ508等位基因和Tay-Sachs病TSD 1278等位基因而产生的LATE-PCR。

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