MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays.
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.
Shapero
Michael H
MH
Affymetrix, Inc., Genotyping Research, 3380 Central Expressway, Santa Clara, CA 95051, USA. michael_shapero@affymetrix.com
Zhang
Jane
J
Loraine
Ann
A
Liu
Weiwei
W
Di
Xiaojun
X
Liu
Guoying
G
Jones
Keith W
KW
D002891:Chromosomes, Human, Pair 21; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers