Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA

Evidentiary items sometimes contain an insufficient quantity of DNA for routine forensic genetic analysis. These so-called low copy number DNA samples (< 100 pg of genomic DNA) often fall below the sensitivity limitations of routine DNA analysis methods. Theoretically, one way of making such intractable samples amenable to analysis would be to increase the number of starting genomes available for subsequent STR (short tandem repeat) analysis by a whole genome amplification strategy (WGA). Although numerous studies employing WGA have focused primarily on clinical applications, few in-depth studies have been conducted to evaluate the potential usefulness of these methods in forensic casework. After an initial evaluation of existing methods, a modified WGA strategy was developed that appears to have utility for low copy number forensic casework specimens. The method employs a slight, but important, modification of the "improved primer extension preamplification PCR" method (I-PEP-PCR), which we term mIPEP (modified-I-PEP-PCR). Complete autosomal STR and Y-STR (Y chromosome short tandem repeat) profiles were routinely obtained with 5 pg of template DNA, which is equivalent to 1-2 diploid cells. Remarkably, partial Y- and autosomal STR profiles were obtained from mIPEP-treated DNA recovered from bloodstains exposed to the outside environment for 1 year whereas non-mIPEP-treated samples did not produce profiles. STR profiles were obtained from contact DNA from single dermal ridge fingerprints when the DNA was subjected to prior mIPEP amplification but not when the mIPEP step was omitted.     

Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification

This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard.     

Whole genome amplification from plant cell colonies of somatic hybrids using strand displacement amplification

The application of strand displacement amplification (SDA) is demonstrated for whole genome amplification from nanograms to micrograms for DNA isolated from small plant cell colonies. Secondary digest amplified fragment length polymorphism (SD-AFLP) analysis confirmed that the amplified genome is a representative of the entire genome. This approach allows the amplification of DNA isolated from small cell colonies of putative somatic hybrids for rapid molecular confirmation of the hybrid status of fusion products.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies

Abstract

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g? WGA using gDNA from two paired cytobrushes (cytobush ‘A’ kept in a cell lysis buffer, and ‘B’ dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.     

Whole gene amplification and protein separation from a few cells

Despite the growing interest to explore untapped microbial gene and protein diversity, no single platform has been able to acquire both gene and protein information from just a few cells. We present a microfluidic system that simultaneously performs on-chip capillary electrophoresis for protein analysis and whole genome amplification (WGA), and we demonstrate this by doing both for the same cohort of cyanobacterial cells. This technology opens avenues for studying protein profiles of precious environmental microbial samples and simultaneously accessing genomic information based on WGA.     

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5 terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3 terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.     

One-step PCR amplification of complete arthropod mitochondrial genomes

A new PCR primer set which enables one-step amplification of complete arthropod mitochondrial genomes was designed from two conserved 16S rDNA regions for the long PCR technique. For this purpose, partial 16S rDNAs amplified with universal primers 16SA and 16SB were newly sequenced from six representative arthropods: Armadillidium vulgare and Macrobrachium nipponense (Crustacea), Anopheles sinensis (Insecta), Lithobius forficatus and Megaphyllum sp. (Myriapoda), and Limulus polyphemus (Chelicerata). The genomic locations of two new primers, HPK16Saa and HPK16Sbb, correspond to positions 13314-13345 and 12951-12984, respectively, in the Drosophila yakuba mitochondrial genome. The usefulness of the primer set was experimentally examined and confirmed with five of the representative arthropods, except for A. vulgare, which has a linearized mitochondrial genome. With this set, therefore, we could easily and rapidly amplify complete mitochondrial genomes with small amounts of arthropod DNA. Although the primers suggested here were examined only with arthropod groups, a possibility of successful application to other invertebrates is very high, since the high degree of sequence conservation is shown on the primer sites in other invertebrates. Thus, this primer set can serve various research fields, such as molecular evolution, population genetics, and molecular phylogenetics based on DNA sequences, RFLP, and gene rearrangement of mitochondrial genomes in arthropods and other invertebrates.     

Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal (AM) fungi

Genetic analysis of arbuscular mycorrhizal (AM) fungi relies on analysis of single spores. The low DNA content makes it difficult to perform large scale molecular analysis. We present the application of Phi29 DNA polymerase mediated strand displacement amplification (SDA) to genomic DNA extracted from single spores of Glomus and Gigaspora species to address this problem. The genome coverage of the SDA process was evaluated by PCR amplification of the beta-tubulin1 gene and part of the rDNA cluster present in AM fungi. The fidelity of SDA was evaluated further by sequencing the Glomus intraradices ITS1 variants to detect the four ITS1 variants previously identified for this fungus.     

PCR amplification following restriction to detect site-specific DNA methylation

A procedure to test for DNA methylation at sites recognized by methylation-sensitive restriction endonucleases is described. The procedure is based on the assumption that the polymerase chain reaction (PCR) will amplify sequences between two primers only if the target DNA is intact after digestion. A carrot (Daucus carota) cell line that is heterozygous for two sequenced alleles ofDc8, a gene which is expressed during the later stages of embryogenesis provided an ideal source of DNA for developing and testing protocols. The promoters of the two alleles differs significantly in length between two sites used for primers, and only one promoter has a GATC (Sau 3A1 orMbo I) site. This allowed development of a protocol where only the sequence lacking the GATC site was amplified to detectable levels following digestion of DNA withMbo I which is insensitive to symmetric methylation with^m4C or^m5C.     

Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.     

Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase

Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.     

2例PCR扩增失败的分析与探讨

根据GenBank报道的序列设计引物,PCR扩增克隆玉米乙醇脱氢酶1(Adh)核基质结合区序列及大鼠脑啡肽基因。扩增的序列电泳分析条带与预期的片段大小基本一致,但测序分析均为非目的片段,为非特异PCR产物,一例为非靶序列间的重复序列配对造成,一例为一侧引物单独引起,重新设计引物,采用巢式PCR获得了目的基因片段。     

DNA amplification fingerprinting: A strategy for genome analysis

A novel strategy to detect genetic differences among organisms, DNA amplification fingerprinting (DAF), uses a thermostable DNA polymerase directed by usually one short (≥5 bp) oligonucleotide primer of arbitrary sequence to amplify short segments of genomic DNA and generate a range of DNA extension products. These products can be analyzed by polyacrylamide gel electrophoresis and silver staining. DAF is rapid and sensitive and is independent of cloning and prior genetic characterization. Here we describe this new methodology, its application to plant genotyping, and its perspectives in DNA fingerprinting and genome mapping.     

Whole genome duplication of intra- and inter-chromosomes in the tomato genome

Whole genome duplication(WGD)events have been proven to occur in the evolutionary history of most angiosperms.Tomato is considered a model species of the Solanaceae family.In this study,we describe the details of the evolutionary process of the tomato genome by detecting collinearity blocks and dating the WGD events on the tree of life by combining two different methods:synonymous substitution rates(Ks)and phylogenetic trees.In total,593 collinearity blocks were discovered out of 12 pseudo-chromosomes constructed. It was evident that chromosome 2 had experienced an intra-chromosomal duplication event.Major inter-chromosomal duplication occurred among all the pseudo-chromosome.We calculated the Ks value of these collinearity blocks.Two peaks of Ks distribution were found,corresponding to two WGD events occurring approximately 36-82 million years ago(MYA)and 148-205 MYA.Additionally, the results of phylogenetic trees suggested that the more recent WGD event may have occurred after the divergence of the rosidasterid clade,but before the major diversification in Solanaceae.The older WGD event was shown to have occurred before the divergence of the rosid-asterid clade and after the divergence of rice-Arabidopsis(monocot-dicot).     

Bst DNA聚合酶大片段在全基因组扩增中的应用

Bst DNA聚合酶大片段作为一种常用的DNA聚合酶,因其独特的特点:能引发链置换反应、高保真、耐高温等,而成为一种重要的DNA多重置换扩增酶。目的:为减少成本,设计一种高产,方便且扩增活性高的Bst DNA聚合酶大片段表达体系;探究该酶应用于胃癌石蜡包埋组织基因组DNA的扩增条件。方法:采用p TWIN1质粒作为载体克隆表达Bst DNA聚合酶大片段,应用几丁质亲和层析柱纯化该酶,使用该酶对人类基因组DNA进行不同温度下扩增,探究其最适反应温度,并据此对胃癌石蜡包埋组织基因组DNA进行扩增。结果:由此得到的Bst DNA聚合酶大片段能运用于胃癌石蜡包埋组织基因组DNA的扩增,扩增效率可达200倍,并能应用于a CGH芯片。结论:扩增得到保真性高,覆盖基因组范围大的DNA扩增产物。该应用与a CGH结合,使得对少量的癌症石蜡包埋组织DNA样本进行全基因组扩增,并进行其基因拷贝数变异研究成为可能。     

Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

Background

Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.

Results

We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792–1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.

Conclusion

Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-856) contains supplementary material, which is available to authorized users.     

Linear nicking endonuclease-mediated strand-displacement DNA amplification

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly.