Robust dosage-PCR for detection of heterozygous chromosomal deletions.
Robust dosage-PCR for detection of heterozygous chromosomal deletions.
复制标题
用于检测杂合染色体缺失的稳健剂量 PCR。
作者：
Qiang Liu;Xuemin Li;Jiesheng Chen;S. Sommer
稳健剂量PCR（RD-PCR）被开发用于检测杂合大缺失，这是常规PCR策略遗漏的一类重要突变。基于PCR的方法可用于区分一个或两个模板拷贝的剂量，但一般应用受到方法的费力性质和/或待分析的每个新基因外显子组所需的优化的限制。RD-PCR依赖于以下两方面的组合：（i）常染色体和X染色体区段的共扩增，以便内部剂量对照可用于任何待分析的区段;以及（ii）稳健的引物设计，其在PCR方案中包括5 '尾和3'序列特异性区域。目标与内部对照段的产率（ROY）比在宽范围内（至少1：1至1：258，相关系数为0.99）与两种输入模板的比成正比。ROY不依赖于基因组DNA的量或在典型条件下扩增的循环数。RD-PCR通过使用内部剂量控制消除了制备和操作步骤中的错误。进行基因剂量的盲法分析，以100%准确度检测人因子IX基因的缺失。前瞻性分析表明，外显子和侧翼剪接点可以分析基因剂量与最小的优化。
Robust dosage-PCR (RD-PCR) was developed to detect heterozygous large deletions, an important class of mutations missed by conventional PCR strategies. PCR-based methods are available for distinguishing between the dosage of one or two template copies, but general application is limited by the laborious nature of the method and/or the optimization required for each new set of gene exons to be analyzed. RD-PCR depends on a combination of (i) co-amplification of an autosomal and an X-chromosomal segment so that internal dosage controls are available for any segment to be analyzed and (ii) a robust primer design that includes a 5'tail and a 3'sequence-specific region in the PCR protocol. The ratio of yields (ROY) of the target to the internal control segment is directly proportional to the ratio of the two input templates over a wide range (at least 1:1 to 1:258 with a correlation coefficient of 0.99). The ROY is not dependent on the amount of genomic DNA or the number of cycles of amplification under typical conditions. RD-PCR eliminates errors in the preparation and manipulation steps by using an internal dosage control. A blinded analysis of gene dosage was performed to detect deletions of the human factor IX gene with 100% accuracy. Prospective analyses demonstrate that exons and flanking splice junctions can be analyzed for gene dosage with minimal optimization.
DOI：
10.1073/pnas.83.24.9373
发表时间：
1986-12-01
影响因子：
11.1
作者：
FREIER, SM;KIERZEK, R;TURNER, DH
通讯作者：
TURNER, DH
影响因子：
2.9
作者：
YOSHITAKE, S;SCHACH, BG;KURACHI, K
通讯作者：
KURACHI, K
DOI：
10.1073/pnas.83.11.3746
发表时间：
1986-06-01
影响因子：
11.1
作者：
BRESLAUER, KJ;FRANK, R;MARKY, LA
通讯作者：
MARKY, LA