Multiplex co-amplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR.
Multiplex co-amplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR.
复制标题
通过长距离 PCR 预扩增后，对 24 个视网膜母细胞瘤基因外显子进行多重共扩增。
作者：
Daizong Li;Jan Vijg
聚合酶链反应（PCR）扩增已成为从DNA或RNA分子的复杂混合物中制备突变分析中DNA模板的首选方法。这一策略适用于小基因或突变热点基因，但当涉及大基因或多基因，且靶片段较多时，基于PCR的突变检测既不劳动也不经济。此外，这种方法受到样本数量的限制。一个典型的例子是识别所有可能的突变，沿着疾病基因的长度分散沿着。处理大量样本的必要性使问题更加复杂。一段时间以来，人们已经知道复杂（高等动物）基因组中的多个靶序列可以同时扩增，即，通过多重PCR。在多重PCR中，不同的DNA片段在相同的条件下在同一反应中共扩增。当目的只是简单地同时扩增多个片段时，有可能克服限制性引物动力学和片段竞争来设计多重系统的最佳条件。然而，当其他约束条件也是相关的，一组条件的设计，允许多路复用大量的基因片段是不平凡的（1）。Chamberlain等人描述了抗肌萎缩蛋白的9个片段的第一个广泛的多重反应。（2）Beggs et al .（3）.这些是例外：大多数多重系统不涉及超过5个扩增子。其明显的原因是，随着每个引物组的增加，允许每个片段达到其退火温度同时避免假扩增产物的允许反应条件变得越来越不灵活。最终，这种灵活性的缺乏是由于基因组序列环境的复杂性，这为非特异性引发提供了充足的机会（1）。当必须根据特定标准选择引物时，如在突变分析中，特殊的约束条件大大降低了多重系统实验设计的灵活性。一个很好的例子是变性梯度凝胶电泳（DGGE），其中引物必须设计成包含具有最佳解链行为的片段。在此类实验中，其中一种引物通常与富含GC的“钳位”序列偶联（4）。在最佳情况下，这将产生一个双结构域结构，GC-钳作为高解链结构域，这种结构将允许检测靶序列中所有可能的突变（5）。通常会发现，
Polymerase chain reaction (PCR) amplification has become themethod of choice for preparing the DNA template in mutationanalysis from complex mixtures of DNA or RNA molecules. Thisstrategy is optimal for small genes or genes with mutational hotspots.However, PCR-based mutation detection is neither labour nor costeffective when large or multiple genes, with many target fragments,are involved. In addition, such an approach is limited by samplequantity. A typical example is the identification of all possiblemutations, scattered along the length of disease genes. The problemis compounded by the necessity of processing large numbers ofsamples.It has been known for some time that multiple target sequencesin complex (higher animal) genomes can be amplified simulta-neously, i.e., by multiplex PCR. In multiplex PCR, different DNAfragments are co-amplified under identical conditions, in the samereaction. When the aim is simply to amplify many fragmentssimultaneously, it is possible to overcome limiting primer kineticsand fragment competition to design optimal conditions for amultiplex system. However, when other constraints are alsopertinent, the design of a set of conditions that allows multiplexingof a large number of gene fragments is not trivial (1). The firstextensive multiplex reactions of nine fragments for the dystrophingene were described by Chamberlain et al . ( 2) and Beggs et al . ( 3).These are exceptions: most multiplex systems do not involve morethan about five amplicons. The obvious reason for this is that witheach primer set added, the permissive reaction conditions allowingeach fragment to reach its annealing temperature while evadingspurious amplification products become increasingly less flexible.Ultimately, this lack of flexibility is due to the complexity of thegenomic sequence environment which allows ample opportunityfor non-specific priming (1).When primers must be selected according to specific criteria,such as in mutation analysis, special constraints greatly lower theflexibility in experimental design of the multiplex system. A goodexample is denaturing gradient gel electrophoresis (DGGE) inwhich primers must be designed to encompass fragments withoptimal melting behaviour. In such experiments, one of theprimers is usually coupled to a GC-rich ‘clamp’ sequence ( 4). Inan optimal situation, this will generate a two-domain structurewith the GC-clamp as the higher melting domain, a configurationwhich will allow the detection of all possible mutations in thetarget sequence (5). It will often turn out that primers positioned
影响因子：
--
作者：
L. Lerman;K. Silverstein
通讯作者：
L. Lerman;K. Silverstein
DOI：
10.1073/pnas.86.1.232
发表时间：
1989-01-01
影响因子：
11.1
作者：
SHEFFIELD, VC;COX, DR;MYERS, RM
通讯作者：
MYERS, RM