Genome amplification of single sperm using multiple displacement amplification.
Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.
Jiang
Zhengwen
Z
Department of Environmental Health, Center for Genome Information, University of Cincinnati College of Medicine 3223 Eden Ave, Cincinnati, OH 45267, USA.
Zhang
Xingqi
X
Deka
Ranjan
R
Jin
Li
L
D023281:Genomics / Q000379:methods; D005838:Genotype; D006801:Humans; D008297:Male; D008540:Meiosis; D018895:Microsatellite Repeats; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013094:Spermatozoa / Q000166:cytology*