Shapero Michael H, Zhang Jane, Loraine Ann, Liu Weiwei, Di Xiaojun, Liu Guoying, Jones Keith W
Affymetrix, Inc., Genotyping Research, 3380 Central Expressway, Santa Clara, CA 95051, USA.
Nucleic Acids Res. 2004 Dec 15;32(22):e181. doi: 10.1093/nar/gnh178.
We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5' end followed by locus-specific sequence at the 3' end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of approximately 95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of approximately 75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.
我们开发了一种用于高度多重单核苷酸多态性（SNP）基因分型的位点特异性DNA靶标制备方法，称为MARA（多重锚定径流扩增）。该方法针对每个SNP使用一条引物，并结合经限制性酶消化、连接了接头的人类基因组DNA。每条引物在5'端由共同序列组成，在3'端由位点特异性序列组成。在产生位点特异性产物的一级反应之后，使用与寡核苷酸和基因组DNA接头序列对应的通用引物对进行二级通用扩增。通过与高密度DNA寡核苷酸阵列杂交实现等位基因区分。最初包含250条引物或750条引物跨九个DNA样本的多重反应表明，250重和750重MARA的平均样本检出率约为95%。我们还评估了用于对人类21号染色体上的SNP进行基因分型的>1000重和4000重MARA。我们已经鉴定出一组SNP，其引物转化率约为75%，在七个DNA样本中显示出平均检出率超过95%且一致性>99%。因此，当与高密度阵列上的等位基因区分相结合时，MARA可能通过在靶标生成过程中允许远超传统多重PCR能力的多重水平，潜在地提高SNP基因分型的通量。
