Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments.
Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments.
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DOI：
10.1093/nar/gni070
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发表时间：
2005-04-28
影响因子：
14.9
通讯作者：
Nilsson M
中科院分区：
文献类型：
--
作者：
Dahl F;Gullberg M;Stenberg J;Landegren U;Nilsson M
我们提出了一种方法，通过使用目标特定的寡核苷酸构建物，即所谓的选择器，通过聚合酶链式反应来特定地选择大量DNA序列进行并行扩增。选择子是具有单链靶互补末端序列的寡核苷酸双链，它们通过一般的序列基序相连。在选择过程中，选择子池与变性的限制性内切酶DNA结合。每个选择子与其各自的靶标杂交，形成单独的环状复合体，通过酶连接共价闭合。通过核外裂解除去非环状片段，对所选片段进行富集化。引入到环状片段中的一般序列允许使用通用引物对并行扩增。该方法避免了与传统的多重聚合酶链式反应相关的扩增伪影，在传统的多重聚合酶链式反应中，每个靶标使用两个引物，从而减少了研究大量DNA序列所需的扩增反应的数量。我们通过对任意一组特定DNA序列进行96-plex扩增，然后与cDNA微阵列杂交，展示了这一过程的特异性、重复性和灵活性。89%的选择子产生了与阵列上预期位置杂交的PCR产物，而几乎没有观察到扩增产物。
We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.
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DOI：
10.1073/pnas.89.13.5847
发表时间：
1992-07-01
影响因子：
11.1
作者：
ZHANG, L;CUI, XF;ARNHEIM, N
通讯作者：
ARNHEIM, N
影响因子：
7
作者：
Hardenbol, P;Yu, FL;Gibbs, RA
通讯作者：
Gibbs, RA
影响因子：
7
作者：
Hacia, JC;Sun, B;Collins, FS
通讯作者：
Collins, FS
影响因子：
46.9
作者：
Hardenbol, P;Banér, J;Davis, RW
通讯作者：
Davis, RW
影响因子：
46.9
作者：
Brenner, S;Johnson, M;Corcoran, K
通讯作者：
Corcoran, K