多重置换扩增——一种新的全基因组扩增技术

全基因组扩增技术用于扩增大量的DNA以满足遗传检测的需要。在高通量的遗传分型中，处理有限的临床样本的基因组DNA，一直是个瓶颈问题。一种新方法—多重置换扩增，能高度忠实的复制整个基因组DNA，扩增出10–100 kb大小的片段，能提供大量均一完整的全基因组序列。MDA是一种简单、有效的方法，非常适用于遗传研究，法医学和临床诊断的需要。     

Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources

The promise of whole genome amplification (WGA) is that genomic DNA (gDNA) quantity will not limit molecular genetic analyses. Multiple displacement amplification (MDA) and the OmniPlex PCR-based WGA protocols were evaluated using 4 and 5 ng of input gDNA from 60 gDNA samples from three tissue sources (mouthwash, buffy coat, and lymphoblast). WGA DNA (wgaDNA) yield and genotyping performance were evaluated using genotypes determined from gDNA and wgaDNA using the AmpFlSTR Identifiler assay and N = 49 TaqMan SNP assays. Short tandem repeat (STR) and SNP genotyping completion and concordance rates were significantly reduced with wgaDNA from all WGA methods compared with gDNA. OmniPlex wgaDNA exhibited a greater reduction in genotyping performance than MDA wgaDNA. Reduced wgaDNA genotyping performance was due to allelic (all protocols) and locus (OmniPlex) amplification bias leading to heterozygote and locus dropout, respectively, and %GC sequence content (%GC) was significantly correlated with TaqMan assay performance. Lymphoblast wgaDNA exhibited higher yield (OmniPlex), buffy coat wgaDNA exhibited higher STR genotyping completion (MDA), whereas mouthwash wgaDNA exhibited higher SNP genotyping discordance (MDA). Genotyping of wgaDNA generated from < or = 5 ng gDNA, e.g., from archaeological, forensic, prenatal diagnostic, or pathology samples, may require additional genotyping validation with gDNA and/or more sophisticated analysis of genotypes incorporating observed reductions in genotyping performance.     

引物延伸预扩增结合简并引物PCR在单细胞比较基因组杂交分析染色体异常中的应用

目的 建立一种可信的单细胞全基因组扩增(whole genome amplification.WGA)技术,结合比较基因组杂交(comparative genomic hybridization,CGH)分析单细胞的染色体拷贝数变化,探讨其在胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用前景.方法 采用引物延伸预扩增结合简并核苷酸引物PCR(primer extension preamplification with degenerate oligonucleotide primed-PCR,PEP-DOP-PCR)的方法,扩增12个已知核型的单细胞标本(包括5个绒毛标本、4个干细胞标本和3个淋巴细胞标本)和4个经PGD检测发现染色体异常的单卵裂球标本,将扩增产物标记红色荧光染料后,与标记绿色荧光染料的正常DNA等量混匀,与正常中期分裂相进行比较基因组杂交分析.同时,应用单纯的简并寡核苷酸引物-PCR(DOP-PCR)扩增10个单细胞DNA,标记后进行CGH分析.比较两种单细胞全基因组扩增方法的扩增效率及随后用于CGH分析染色体拷贝数时的准确性.结果 所有的单细胞采用PEP-DOP-PCR扩增时,均能获得稳定均匀的PCR产物,片段大小范围在100～1000 bp之间,集中分布于400 bp左右的区域,CGH分析结果显示染色体拷贝数变化与其它技术检测的结果一致.而10个单纯的DOP-PCR扩增只有6个标本成功,扩增产物进行CGH分析时,杂交信号不均匀,有2个显示与其它技术分析的结果不一致.结论 PEP-DOP-PCR技术能有效地扩增单细胞的全基因组DNA,其扩增产物可应用CGH技术成功检测单个细胞的染色体拷贝数变化,而单纯的DOP-PCR技术易于出现扩增失败、扩增产物杂交后信号不均一的缺点.PEP-DOP-PCR全基因组扩增结合CGH技术在胚胎植入前遗传学诊断中有良好的应用前景.     

两种全基因组扩增方法用于微量检材STR基因座分型比较

目的 应用多重置换扩增(MDA)技术和改进型扩增前引物延伸（IPEP）技术对法医学微量DNA进行全基因组扩增，比较两种方法的STR分型效果及法医学应用价值。 方法 用MDA、IPEP方法分别对不同模板量DNA进行扩增，扩增产物用实时荧光定量PCR技术定量、用AmpFLSTR® Identifiler®试剂盒检测基因型。 结果 MDA方法可对模板DNA增加103～106倍，IPEP方法可增加25～310倍。为获得完整准确的分型结果，MDA最低需1ng基因组DNA，IPEP最低需0.05ng基因组DNA。当基因组DNA为0.01ng～0.1ng时，IPEP产物、MDA产物的平均基因座检出数均高于未经全基因组扩增的DNA，其中IPEP产物的平均基因座检出数高于MDA产物。 结论 MDA方法、IPEP方法均可提高微量检材的STR分型效果。MDA方法的产量高于IPEP方法；IPEP方法的灵敏度高于MDA方法，且对微量DNA的STR分型效果优于MDA方法，因此更适于法医学痕量DNA检测。     

多重置换扩增在植入前遗传学诊断中的应用及展望

多重置换扩增是一种新兴的全基因组扩增技术,能对单个细胞进行全基因扩增,产生大量的优质DNA,具有高扩增效率和高保真性等特点.多重置换扩增联合常规PCR已被成功用于植入前遗传学诊断,进一步扩展了后者的应用范围.     

Optimization and evaluation of single-cell whole-genome multiple displacement amplification

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.     

A New Workflow for Whole‐Genome Sequencing of Single Human Cells

Unbiased amplification of the whole‐genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter‐linker PCR‐based WGA method with second‐generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy‐number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR‐based WGA approach. Sequencing with paired‐end reads allowed genome‐wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.     

Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R

The identification of common genetic variants such as single nucleotide polymorphisms (SNPs) in the human genome has become central in human population genetics and evolution studies, as well as in the study of the genetic basis of complex traits and diseases. Crucial for the accurate identification of genetic variants is the availability of high quality genomic DNA (gDNA). Since popular sources of gDNA (buccal cells, lymphocytes, hair bulb) often do not yield sufficient quantities of DNA for molecular genetic applications, whole genome amplification methods have recently been introduced to generate a renewable source of double-stranded linear DNA. Here, we assess the fidelity of one method, multiple displacement amplification (MDA), which utilizes bacteriophage Phi29 DNA polymerase to generate amplified DNA from an original source of gDNA, in a representative SNP discovery and genetic association study at the melanocortin 1 receptor (MC1R) locus, a highly polymorphic gene in humans involved in skin and hair pigmentation. We observed that MDA has high fidelity for novel SNP discovery and can be a valuable tool in generating a potentially indefinite source of DNA. However, we observed an allele amplification bias that causes genotype miscalls at heterozygous sites. At loci with multiple polymorphic sites in linkage disequilibrium, such as at MC1R, this bias can create a significant number of heterozygote genotype errors that subsequently misrepresents haplotypes.     

Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation

For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template.     

Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms

Highly accurate and high-throughput SNP genotyping platforms are increasingly popular but the performance of suboptimal DNA samples remains unclear. The aim of our study was to determine the best platform, amplification technique, and loading concentration to maximize genotype accuracy and call rate using degraded samples. We amplified high-molecular weight genomic DNA samples recently extracted from whole blood and degraded DNA samples extracted from 50-year-old patient sera. Two whole-genome amplification (WGA) methodologies were used: an isothermal multiple displacement amplification method (MDA) and a fragmentation-PCR-based method (GenomePlex [GPLEX]; Sigma-Aldrich, St. Louis, MO). Duplicate runs were performed on genome-wide dense SNP arrays (Nsp-Mendel; Affymetrix) and custom SNP platforms based on molecular inversion probes (Targeted Genotyping [TG]; Affymetrix) and BeadArray technology (Golden Gate [GG]; Illumina). Miscalls and no-calls on Mendel arrays were correlated with each other, with confidence scores from the Bayesian calling algorithm, and with average probe intensity. Degraded DNA amplified with MDA gave low call rates and concordance across all platforms at standard loading concentrations. The call rate with MDA on GG was improved when a 5 x concentration of amplified DNA was used. The GPLEX amplification gave high call rate and concordance for degraded DNA at standard and higher loading concentrations on both TG and GG platforms. Based on these analyses, after standard filtering for SNP and sample performance, we were able to achieve a mean call rate of 99.7% and concordance 99.7% using degraded samples amplified by GPLEX on GG technology at 2 x loading concentration. These findings may be useful for investigators planning case-control association studies with patient samples of suboptimal quality.     

Sample selection algorithm to improve quality of genotyping from plasma-derived DNA: to separate the wheat from the chaff

Plasma and serum samples were often the only biological material collected for earlier epidemiological studies. These studies have a huge informative content, especially due to their long follow-up and would be an invaluable treasure for genetic investigations. However, often no banked DNA is available. To use the small amounts of DNA present in plasma, in a first step, we applied magnetic bead technology to extract this DNA, followed by a whole-genome amplification (WGA) using phi29-polymerase. We assembled 88 sample pairs, each consisting of WGA plasma DNA and the corresponding whole-blood DNA. We genotyped nine highly polymorphic short tandem repeats (STRs) and 23 SNPs in both DNA sources. The average within-pair discordance was 3.8% for SNPs and 15.9% for STR genotypes, respectively. We developed an algorithm based on one-half of the sample pairs and validated on the other one-half to identify the samples with high WGA plasma DNA quality to assure low genotyping error and to exclude plasma DNA samples with insufficient quality: excluding samples showing homozygosity at five or more of the nine STR loci yielded exclusion of 22.7% of all samples and decreased average discordance for STR and SNP markers to 3.92% and 0.63%, respectively. For SNPs, this is very close to the error observed for genomic DNA in many laboratories. Our workflow and sample selection algorithm offers new opportunities to recover reliable DNA from stored plasma material. This algorithm is superior to testing the amount of input DNA.     

微阵列比较基因组杂交技术及其应用

【摘要】基因组DNA拷贝数变化在许多人类疾病如肿瘤、遗传性疾病的发生发展中起重要作用。经典的比较基因组杂交技术由于分辨率低,只能检测较大的拷贝数变化。微阵列比较基因组杂交技术具有高分辨率、高灵敏度、高通量和自动化等优点,能准确检测单拷贝的缺失、复制和扩增,并把结果直接定位于基因组上,为肿瘤、遗传性疾病及正常人群基因组DNA拷贝数变化的研究提供了一种有效的方法。     

Unbiased whole-genome amplification directly from clinical samples

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods.     

Evaluation of 3 methods of whole-genome amplification for subsequent metaphase comparative genomic hybridization.

A common aim in cancer research is to investigate mechanisms of malignant progression by genetic analysis of key stages, including pre-malignancy, microinvasion, and micrometastases. As such lesions are small and require microdissection from clinical samples, the amount of DNA that can be recovered is limited and frequently inadequate for commonly used techniques of genomic analysis, such as comparative genomic hybridization (CGH). There is a critical requirement for techniques of whole-genome amplification that minimize representation bias in the amplified sample. Several techniques have been described, although their relative suitability for CGH has not been examined adequately. Here we compare the abilities of degenerate oligonucleotide-primed PCR (DOP-PCR), multiple-strand displacement amplification (MDA), and balanced PCR accurately to amplify limited amounts of template DNA for use in CGH. Amplification by DOP-PCR and MDA, but not balanced PCR faithfully preserved the original genomic content following amplification, as evidenced by generally concordant CGH copy number karyograms. Whereas the amplification products of DOP-PCR were immediately available for labeling and hybridization, the products of MDA required a further digestion step to produce optimal-sized probes for CGH. Moreover, MDA was less reliable overall than DOP-PCR at the lowest starting amount of 10 pg of template DNA. We conclude that DOP-PCR is the method of choice for whole-genome amplification of minute quantities of DNA to enable global genomic analysis to be performed on limited clinical samples.     

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.     

利用基因芯片对少量细胞进行非整倍体检测的影响因素分析

目的 评估利用微阵列-比较基因组杂交技术检测少量细胞非整倍体的准确率及相关影响因素.方法 结合10K 2.0单核苷酸多态性(SNP)基因分型芯片平台与多重置换扩增技术(MDA),计算并比较扩增模板为1～10个细胞时各染色体拷贝数分析的准确率,评估影响芯片平台的拷贝数准确率的有关因素及其对染色体拷贝数异常的实际分辨率.结果 使用MDA-DNA作参照时,拷贝数分析的准确率[(79.3±2.9)%～(100.0±1.7)%]高于使用gDNA作参照时的准确率[(66.7±3.4)%～(89.5±3.3)%](P<0.001).随着模板增加至10细胞,芯片可在1 M平滑化处理的同时获得94%的分析准确率.对于单细胞MDA产物,缺失型非整倍体具有比获得型非整倍体更高的分析准确率[1C组(71.9±4.1)%～(95.5～2.0)%;1C-sDel-4组(81.4±3.7)%～(99.6±2.8)%],各组间差异均有统计学意义(P<0.01).结论 10K 2.0 SNP基因分型芯片平台结合多重置换扩增技术可有效对少量细胞进行非整倍体检测,选择MDA-DNA作为参照是提高分析准确率的最关键因素,而增加细胞模板与提升分析中的平滑化参数(即降低芯片的分辨率要求)也有助于改善拷贝数准确率,在同样的条件下,芯片更容易准确检出缺失型非整倍体.     

全基因组扩增技术最新进展及其法医学应用现状

微量模板DNA的检测，是很多领域迫切需要解决的一个难题。因为其DNA量不足，用现有的技术手段常无法检测成功。全基因组扩增技术可对非常微量的DNA进行均衡的扩增而获得大量的DNA，故被认为是目前解决这一难题的一种基本方法，已被广泛用于法医学、单细胞遗传病的诊断及疾病基因的分析等领域研究，并取得了良好的效果。本文对这一技术的最新研究进展及其在法医学方面的应用现状做一综述。     

Preimplantation testing: Transition from genetic to genomic diagnosis

Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization (IVF) prior to initiating a pregnancy. Traditional genetic methods for preimplantation genetic diagnosis (PGD) examine distinct parts of an individual genome, require the development of a custom assay for every patient family, and are time consuming and inefficient. In the last decade technologies for whole-genome amplification (WGA) from single cells have led to innovative strategies for preimplantation testing. Applications of WGA technology can lead to a universal approach that uses single-nucleotide polymorphisms (SNPs) and mutations across the entire genome for the analysis. Single-cell WGA by multiple displacement amplification has enabled a linkage approach to PGD known as “preimplantation genetic haplotyping”, as well as microarray-based techniques for preimplantation diagnosis. The use of microarrays in preimplantation diagnosis has provided genome-wide testing for gains or losses of single chromosomes (aneuploidies) or chromosomal segments. Properly designed randomized controlled trials are, however, needed to determine whether these new technologies improve IVF outcomes by increasing implantation rates and decreasing miscarriage rates. In genotype analysis of single cells, allele dropout occurs frequently at heterozygous loci. Preimplantation testing of multiple cells biopsied from blastocysts, however, can reduce allele dropout rates and increase the accuracy of genotyping, but it allows less time for PGD. Future development of fast SNP microarrays will enable a universal preimplantation testing for aneuploidies, single-gene disorders and unbalanced translocations within the time frame of an IVF cycle.     

Multiple displacement amplification,a powerful tool for molecular genetic analysis of powdery mildew fungi

Powdery mildew fungi (Erysiphales) are probably the largest group of plant pathogens that remain uncharacterized from genetic and molecular points of view, with the only exception of the powdery mildew of cereals, Blumeria graminis. Their nature as obligate biotrophic parasites and consequent inability to grow on culture media has significantly hampered research. A common bottleneck to the molecular genetic analysis of powdery mildew fungi is the availability of genomic DNA of suitable quality and in sufficient quantity. The so-called whole genome amplification technology has the potential to overcome this limitation. Here we present the application of phi29 DNA polymerase-mediated multiple displacement amplification (MDA) to amplify the whole genome of Podosphaera fusca, the main causal agent of powdery mildew in cucurbits, to address this problem. The genome coverage and fidelity of the MDA process was evaluated by PCR amplification and sequencing of two genetics markers: the nuclear rDNA internal transcribed spacer (ITS) regions and the mitochondrial cytochrome b gene (CYTB). Our results show that MDA is a valuable tool for molecular genetic analysis of powdery mildew fungi that can be used for a number of downstream applications in different fields, such as epidemiology and population genetics or systematics.     

Molecular typing of human leukocyte antigen and related polymorphisms following whole genome amplification

Reliable, high-resolution genotyping of human leukocyte antigen (HLA) polymorphisms is often compromised by DNA samples of suboptimal quality or limited quantity. We tested the feasibility of molecular typing for variants at HLA and neighboring loci using whole genome amplification (WGA) strategy facilitated by the Phi29 DNA polymerase. With little (5-100 ng) starting genomic DNA of varying quality and source materials, WGA was deemed successful in 167 of 169 DNA from 47 cell lines, 100 European Americans, and 22 native Africans. The Phi29-processed DNA provided adequate templates for polymerase chain reaction (PCR)-based analyses of several HLA (A, B, C, DRB1, and DQB1) and related loci (HFE, MICA, and 10 microsatellites) in the 6p24.3-6p21.3 region, with PCR amplicons ranging from 92 to 2200 bp. Five different genotyping techniques resolved and confirmed 364 genotypes when both original and Phi29-processed DNA worked in PCRs. General population genetic analyses provided additional evidence that WGA may represent a reliable and simple approach to securing ample genomic DNA for typing HLA, MICA, and related variants.