Genome amplification of single sperm using multiple displacement amplification

Department of Environmental Health, Center for Genome Information, University of Cincinnati College of Medicine 3223 Eden Ave, Cincinnati, OH 45267, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(10):e91. DOI: 10.1093/nar/gni089
Source: PubMed

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.

1 Follower
  • Source
    • "PCR-based methodology has been used frequently to study haplotypes and recombination events in single sperm cells e.g. (Hubert et al. 1994), and MDA has enabled extensive genotyping (Jiang et al. 2005). Recently, genome-wide analysis of individual sperm cells after MDA was used to assess recombination activity and de novo mutation rates (Wang et al. 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: There is increasing evidence that the phenotypic effects of genomic sequence variants are best understood in terms of variant haplotypes rather than as isolated polymorphisms. Haplotype analysis is also critically important for uncovering population histories, and for the study of evolutionary genetics. Although the sequencing of individual human genomes to reveal personal collections of sequence variants is now well established, there has been slower progress in the phasing of these variants into pairs of haplotypes along each pair of chromosomes. Here, we have developed a distinct approach to haplotyping that can yield chromosome-length haplotypes, including the vast majority of heterozygous SNPs in an individual human genome. This approach exploits the haploid nature of sperm cells, and employs a combination of genotyping and low-coverage sequencing on a short-read platform. In addition to generating chromosome-length haplotypes, the approach can directly identify recombination events (averaging 1.1 per chromosome) with a median resolution of less than 100 kb.
    Genome Research 01/2013; 23(5). DOI:10.1101/gr.144600.112 · 13.85 Impact Factor
  • Source
    • "In principle, WGA can amplify DNA template in an unbiased manner with complete genome coverage and high accuracy in an isothermal reaction [5] [6]. WGA applications have promise for clinical samples [7] [8], single cells [9] [10] [11] [12] [13], formalin-fixed, paraffinembedded samples [14–18], post-mortem brain samples [19], and archived samples [20,21]. WGA has also successfully amplified DNA for STR genetic profiling from LT or degraded DNA samples, including touch DNA obtained from single dermal ridge fingerprints [22] [23] [24] [25] [26], for mtDNA sequencing [27], and for SNP genotyping [4,16,28–30]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework.
    Forensic Science International: Genetics 05/2011; 6(2):185-90. DOI:10.1016/j.fsigen.2011.04.011 · 3.20 Impact Factor
  • Source
    • "have pointed out its great potential for producing high-coverage and high-yield WGA products from single sperm cells. They estimated that 76% of the loci can be amplified at least 2500-fold from single sperm DNA, and it can be further amplified >200-fold by a second-round MDA (Jiang et al., 2005 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation, improving the chance of conception for patients at high risk of transmitting specific inherited disorders. This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s. Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD, but there are some inevitable shortcomings limiting the scope of genetic diagnosis. Fortunately, different whole genome amplification (WGA) techniques have been developed to overcome these problems. Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed. Moreover, WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis. In this review, we will focus on the currently available WGA techniques and their applications, as well as the new technical trends from WGA products.
    Journal of Zhejiang University SCIENCE B 01/2011; 12(1):1-11. DOI:10.1631/jzus.B1000196 · 1.29 Impact Factor
Show more


Available from