Genome amplification of single sperm using multiple displacement amplification - PubMed (original) (raw)

Genome amplification of single sperm using multiple displacement amplification

Zhengwen Jiang et al. Nucleic Acids Res. 2005.

Abstract

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.

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Figures

Figure 1

Figure 1

PCR amplification of the three fragments of the TOP1, P53 and CYP1A2 genes in MDA products of 16 sperm aliquots treated using two different lysis methods. The fragments are 1080 bp in length for TOP1, 643 bp for P53 and 550 bp for CYP1A2. Eight aliquots from the dilution of 3 cells/3 μl were lysed on ice and the other eight were lysed at 65°C.

Figure 2

Figure 2

PCR amplification of the three fragments of the TOP1, P53 and CYP1A2 genes in MDA products of S01–S16 samples. The fragments are 1080 bp in length for TOP1, 643 bp for P53 and 550 bp for CYP1A2. S01–S16 indicate MDA products from 16 sperm aliquots. S16MDA2 is the second-round MDA product using 1 μl of 1/10C0 S16 MDA product as resource DNA. BLANK here is the MDA product of the negative control, in which 3 μl PBS buffer was added instead of sperm aliquot.

Figure 3

Figure 3

PCR amplification of 12 fragments using 1 μl of 1/5C0 or 1/50C0 MDA products as templates. The amplification result is indicated with a black square for successful amplification in 1/50C0, a gray square for successful amplification in 1/5C0 and a framed F for no amplification in both.

Figure 4

Figure 4

MDA products were useful for a variety of genetic applications, including sequencing and SNP and STR analysis. (A) A mutation at 45803241 of NT_010194.16 is marked by red arrows in the S14 MDA product. (B) Sequencing traces for rs1048943 (marked by red arrows) and its flanking 10 nt in genomic DNA, S14, S06 and S16 MDA products. S06 has two alleles, although the C allele was much preferred for amplification. (C) The electropherogram for the D10S547 locus in genomic DNA, S14, S04 and S05 samples displayed by GeneMapper V3.0. Their genotypes were displayed in Table 5. S14 was observed to have two alleles (244 and 254), although allele 254 showed to be preferentially amplified. The orange peak is a 250 bp marker. (D) The interface for the identification of genotypes at rs1535721 locus by Taqman assay using the ABI7900. The green one is for genomic DNA sample, blue for S05, S13 and S15, and red for S06, S08, S14, S16 and S16MDA2. × is for S04, for which the amplification failed.

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