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。     

全基因组扩增技术的研究进展

全基因组扩增可增加微量DNA分析的遗传信息量，为实现微量DNA多基因位点分析和重复检测提供了可能，目前已应用于植入前遗传学诊断，母体外周血胎儿细胞产前遗传学分析。及肿瘤基因分析等领域研究，是一项非常有价值的技术。     

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。     

全基因组扩增技术的研究进展

全基因组扩增可增加微量 DNA分析的遗传信息量 ,为实现微量 DNA多基因位点分析和重复检测提供了可能 ,目前已应用于植入前遗传学诊断、母体外周血胎儿细胞产前遗传学分析 ,及肿瘤基因分析等领域研究 ,是一项非常有价值的技术。     

中国成都汉族群体线粒体DNA控制区序列多态性研究

目的　检测并分析中国成都汉族群体线粒体 DNA控制区多态性 ,为法医学应用提供基础数据。方法　应用 PCR扩增和直接测序方法检测 10 0个不相关中国成都汉族群体线粒体 DNA控制区中高变区 、 的序列多态性。结果　检测高变区 中 4 0 4个核苷酸和高变区 中 379个核苷酸的序列 ,在 区和 区分别检测到 92和 5 0个变异点 ,与 Anderson标准序列比较 ,共检测到 97种单倍型。偶合概率分别为1.84 %与 1.94 % ,两者合并后为 1.18%。结论　提示线粒体 DNA控制区 DNA序列多态性在法医学领域中具有较高的应用价值。     

PCR技术的研究进展与临床应用

聚合酶链反应 (Polymerase Chain Reactionm ,PCR)是一项体外基因扩增技术 ,1985年美国 PE公司人类遗传研究室 Mullis等人发明了该项技术 ,Saiki等首先应用于镰状红细胞贫血的产前诊断〔1〕 ,但由于操作方法繁琐未能全面推广应用。直到 1988年耐热 DNA聚合酶 (Taq酶 )的发现和应用〔2〕 ,使 PCR技术变得极为简单 ,才被迅速应用于分子生物学、生物工程、医学、法医学领域 ,也使人类疾病的基因诊断进入了临床实用阶段。历经十余年 ,PCR技术已由当初只能对样品中的 DNA和 RNA定性分析发展到能够进行定量分析。本文对 PCR技术的研究…     

聚合酶链反应在病理诊断应用中的体会

聚合酶链反应(polymerase chain reaction,PCR)技术是分子生物学领域近年来发展的一种快速的特定DNA片断体外扩增法,并很快被各学科广泛应用,但目前这一技术在病理诊断中应用尚不多。我们应用PCR技术对     

DNA分析技术在法医学中的应用及进展

DNA分析技术在许多研究和应用领域发挥重要的作用，尤其在法医学领域具有重要意义。本文对DNA指纹技术、STR、mtDNA测序、SNP等主要DNA分析技术的应用和发展，以及建立DNA数据库的必要性和应用价值进行了系统的阐述。     

免疫-PCR技术进展

免疫-PCR结合了抗原抗体反应的特异性和PCR的高敏感性,是一种极为敏感的抗原检测技术,并适合于各种微量抗原的检测。免疫-PCR的关键在于形成抗体-标记DNA偶联物,作为桥梁连接免疫反应的特异性和PCR的高扩增能力。本文简述了免疫-PCR的基本流程以及抗体与标记DNA相偶联、PCR产物检测方法的进展。     

PCR固相分析法

PCR固相分析法是近年来发展起来的新技术,其主要创新是使DNA扩增产物结合在固相载体上直接进行分析和检测。与传统的凝胶电泳等法比较,本法更为简单实用。目前已在病原体的基因诊断、DNA顺序测定、扩增DNA片段的纯化及定点诱变等领域的研究中显示出良好的应用潜力。本文简要综述这一技术的基本原理及其应用。     

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.     

Application of DNA markers to clinical genetics

Summary DNA technology using DNA sequence polymorphisms has brought a new system to the fields of medicine and forensic science, especially for the studies of genetic diseases and tumor suppressor genes, and for identification of individuals for forensic purposes. Linkage analysis based on segregation of polymorphic alleles in affected families has contributed to identification of many genetic diseases. We isolated a large number of polymorphic DNA markers, called VNTR (variable number of tandem repeat) markers and identified the APC gene that is responsible for familial adenomatous polyposis (FAP) by means of a so-called positional cloning and characterized germline and somatic mutations of the APC gene in colorectal cancer patients. In addition, we have applied genetic information during colorectal carcinogenesis to sensitive diagnosis of lymph-node metastasis of colorectal cancer.     

Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells

Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.     

Application of forensic DNA testing in the legal system

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.     

Multiple displacement amplification to create a long-lasting source of DNA for genetic studies

In many situations there may not be sufficient DNA collected from patient or population cohorts to meet the requirements of genome-wide analysis of SNPs, genomic copy number polymorphisms, or acquired copy number alternations. When the amount of available DNA for genotype analysis is limited, high performance whole-genome amplification (WGA) represents a new development in genetic analysis. It is especially useful for analysis of DNA extracted from stored histology slides, tissue samples, buccal swabs, or blood stains collected on filter paper. The multiple displacement amplification (MDA) method, which relies on isothermal amplification using the DNA polymerase of the bacteriophage phi29, is a recently developed technique for high performance WGA. This review addresses new trends in the technical performance of MDA and its applications to genetic analyses. The main challenge of WGA methods is to obtain balanced and faithful replication of all chromosomal regions without the loss of or preferential amplification of any genomic loci or allele. In multiple comparisons to other WGA methods, MDA appears to be most reliable for genotyping, with the most favorable call rates, best genomic coverage, and lowest amplification bias.     

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.     

Assessment of fidelity and utility of the whole-genome amplification for the clinical tests offered in a histocompatibility and immunogenetics laboratory

Increasing emphasis on the use of molecular tests in a histocompatibility and immunogenetics laboratory (HIL) poses a potential problem of lack of sufficient DNA to perform multiple genetic analyses. In this study, we report the feasibility, fidelity and utility of multiple displacement amplification (MDA) method to perform whole-genome amplification (WGA) to generate DNA specimens that can be analyzed by multiple molecular techniques and can be used for different clinical tests offered by an HIL. The MDA-generated DNA when compared with the native DNA showed 100% congruency in genotyping of 37 genes/loci using multiple downstream molecular techniques: sequence-based typing and sequence-specific primer-based typing for 5 human leukocyte antigen (HLA) class I and II genes (HLA-A, B, C, DRB1 and DQB1), luminex-based sequence-specific oligonucleotide (SSO) genotyping for a panel of 16 killer immunoglobulin-like receptor (KIR) genes and automated fragment size analysis for a panel of 15 short tandem repeat (STR) loci and amelogenin gene. For post-allogeneic hematopoietic cell transplantation (HCT) chimerism analysis, MDA-generated DNA appeared useful for enriching pre-transplant DNA but not for enriching post-transplant chimeric DNA. Overall, our results show that MDA-based WGA could generate DNA of high yield and fidelity that can be used for various clinical tests and research purposes.     

Use of dried blood spots for the determination of genetic variation of interleukin-10, killer immunoglobulin-like receptor and HLA class I genes

Optimal methods for using dried blood spots (DBSs) for population genetics-based studies have not been well established. Using DBS stored for 8 years from 21 pregnant South African women, we evaluated three methods of gDNA extraction with and without whole-genome amplification (WGA) to characterize immune-related genes: interleukin-10 (IL-10), killer immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I. We found that the QIAamp DNA mini kit yielded the highest gDNA quality (P< 0.05; Wilcoxon signed rank test) with sufficient yield for subsequent analyses. In contrast, we found that WGA was not reliable for sequence-specific primer polymerase chain reaction (SSP-PCR) analysis of KIR2DL1, KIR2DS1, KIR2DL5 and KIR2DL3 or high-resolution HLA genotyping using a sequence-based approach. We speculate that unequal template amplification by WGA underrepresents gene repertoires determined by sequence-based approaches.