Callow Matthew J, Drmanac Snezana, Drmanac Radoje
Callida Genomics Inc., 675 Almanor Avenue, Sunnyvale, CA 94085, USA.
Nucleic Acids Res. 2004 Jan 28;32(2):e21. doi: 10.1093/nar/gnh019.
There is a rapidly developing need for new technologies to amplify millions of different targets from genomic DNA for high throughput genotyping and population gene-sequencing from diverse species. Here we describe a novel approach for the specific selection and amplification of genomic DNA fragments of interest that eliminates the need for costly and time consuming synthesis and testing of potentially millions of amplicon-specific primers. This technique relies upon Type IIs restriction enzyme digestion of genomic DNA and ligation of the fragments to double-sided adapters to form closed-circular DNA molecules. The novel use of double-sided adapters, assembled through the combinatorial use of two small universal sets of oligonucleotide building blocks, provides greater selection capacity by utilizing both sides of the adapter in a sequence-specific ligation event. As demonstrated, formation of circular structures results in protection of the desired molecules from nuclease treatment and enables a level of selectivity high enough to isolate single, or multiple, pre-defined fragments from the human genome when digested at over five million sites. Priming sites incorporated into the adapter allows the utilization of a common pair of primers for the amplification of any adapter-captured DNA fragment of interest.
对于新技术的需求正在迅速增长，这些技术用于从基因组DNA中扩增数百万个不同的靶标，以进行高通量基因分型和来自不同物种的群体基因测序。在此，我们描述了一种用于特异性选择和扩增感兴趣的基因组DNA片段的新方法，该方法无需对潜在的数百万个扩增子特异性引物进行昂贵且耗时的合成和测试。该技术依赖于对基因组DNA进行IIs型限制性内切酶消化，并将片段连接到双面衔接子上以形成闭环DNA分子。通过组合使用两组小的通用寡核苷酸构建块组装而成的双面衔接子的新颖应用，通过在序列特异性连接事件中利用衔接子的两侧提供了更大的选择能力。如所证明的，环状结构的形成导致所需分子免受核酸酶处理的影响，并使得当在超过五百万个位点进行消化时，能够具有足够高的选择性水平以从人类基因组中分离单个或多个预定义片段。掺入衔接子中的引物位点允许使用一对通用引物来扩增任何感兴趣的衔接子捕获的DNA片段。
