微阵列-比较基因组杂交技术及其在肿瘤学中的应用

人类肿瘤的发生和发展常与非随机性染色体异常(包括基因组缺失、获得、扩增和重排等)有关。比较基因组杂交(comparative genomic hybridization，CGH)技术可以快速全面地分析肿瘤组织DNA拷贝数的变化，并在中期染色体上定位，对于肿瘤研究具有重要意义。但是，CGH至今未应用于临床，一个主要的障碍是使用中期染色体铺片作为杂交     

比较基因组杂交技术的应用

肿瘤发生和发育畸形与染色体的不平衡有关,包括染色体的扩增和缺失.常规的比较基因组杂交技术(conventional comparative genomic hybridization)由于能在一次试验中掌握整个基因组DNA拷贝数的变化并可以将此改变准确的定位于染色体,因而在探讨肿瘤的发生和生长发育畸形机理的研究方面得到了广泛的应用.在此基础上发展起来的微阵列比较基因组杂交技术(microarray CGH/ array-based CGH),为精确、定量地研究人类基因组微缺失和微扩增及其定位提供了有力工具.本文综述了CGH和CGHa 技术的原理及应用.     

比较基因组杂交在胃肠道肿瘤中的应用

比较基因组杂交 (CGH)是一种将消减杂交和荧光原位杂交 (FISH)相结合的分子细胞遗传学技术 ,可在全部染色体区带上检测基因组间 DNA拷贝数变化并定位。本文综述了 CGH检测胃肠道肿瘤的研究结果及其应用     

比较基因组杂交在结直肠癌中的应用

比较基因组杂交 (CGH)是一种将消减杂交和荧光原位杂交 (FISH)相结合的分子细胞遗传学技术 ,可在全部染色体区带上检测基因组间DNA的拷贝数变化并定位。本文综述了CGH检测结直肠癌中的研究进展及其应用。     

单细胞引物延伸预扩增分析多个基因

目的 建立一种单细胞（individual cell)水平引物延伸预扩增（primer extension preamplification,PEP)方法,利用单个细胞同时分析多个基因。方法 采用10bp的随机引物先对单细胞的基因组预扩增,取其产物5μl分别对SRY、ZP3、RhCE、RhD、FⅧ5个基因进行套式PCR扩增。结果 单细胞PEP后的产物能同时扩增5个基因;对4种已知基因型的白细胞进行诊断,正确度为95．6％。结论 PEP技术可以在单细胞水平分析5个基因,可用于胚胎种植前诊断及无创性产前诊断。     

寡核苷酸阵列比较基因组杂交技术在肿瘤研究中作用

人类肿瘤的发生发展具有遗传相关性[1],几乎所有的肿瘤存在基因组DNA拷贝数异常,这些异常与原癌基因的扩增和抑癌基因的缺失密切相关.因此,利用先进的分子遗传学新技术,研究肿瘤组织中基因组DNA异常,已成为人类了解肿瘤发生机制的重心.1992年,Kallioniemi等首次提出比较基因组杂交(CGH)技术,能对染色体上DNA序列进行检测并定位.     

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。     

用比较基因组杂交技术(CGH)检测肾癌染色体畸变

目的应用比较基因组杂交技术(CGH)分析原发性肾癌肿瘤组织中染色体异常变化,探讨肾癌细胞遗传物质的改变,揭示肾癌发生发展的内在本质及其与临床特征之间的关系。方法采用CGH技术对12例肾癌组织提取的全基因组DNA进行检测,以了解肾癌全基因组的变化。结果CGH技术检测的12例肾癌标本中均有染色体的畸变(扩增和/或缺失),常见的扩增区是1p、4p、5q、7p、9p、16p,常见的缺失区是3q、4q、6q、9q、14q、18q。结论原发性肾癌存在广泛的遗传物质不平衡现象,肾癌细胞染色体扩增和/或缺失可能是肾癌发生发展的基础。     

微阵列比较基因组杂交技术在肿瘤研究中作用

人类肿瘤的研究重心经历了从单基因水平到染色体、多基因水平的转变。许多人类肿瘤与发育异常疾病一样以基因组DNA拷贝数变化为特点。在癌症中,不利于肿瘤生长的基因（抑癌基因）可能存在于DNA拷贝数减少的区域;而有利于肿瘤发生、发展的基因（癌基因）可能存在于DNA拷贝数增加的区域。1992年,Kallioniemi等。首次描述了比较基因组杂交（CGH）技术。其基本原理是：分别用不同的荧光标记体系标记来自待检组织和正常组织的全基因组DNA,[第一段]     

应用比较基因组杂交技术进行畸形胎儿的染色体核型分析

目的应用比较基因组杂交（Comparative Genomic Hybridization,CGH）方法,快速进行畸形胎儿的染色体核型分析。方法选取畸形胎儿标本11例,采用Tiangen DNA抽提试剂盒提取羊水或脐血基因组DNA,Vysis缺口平移试剂盒标记待测DNA和对照DNA,同时将待测DNA和对照DNA的荧光颜色互换,即进行荧光互换CGH,最后根据绿红两种信号的比值制作CGH拷贝数核型模式图。结果11例畸形胎儿的样本均成功的利用荧光互换CGH方法进行了分析、确认,平均用时3.5天。其中1例CGH核型为Dup21,1例CGH核型为Del2p24-pter,Dup12p13,1例CGH核型为Del1p33-pter,Del22q11-12。结论荧光互换CGH方法快速进行畸形胎儿染色体的核型分析具有可行性。     

Improved DOP-PCR-based representational whole-genome amplification using quantitative real-time PCR.

In many cases, only a minute amount of partially degraded genomic DNA can be extracted from archived clinical samples. Diverse whole-genome amplification methods are applied to provide sufficient amount of DNA for comparative genome hybridization, single-nucleotide polymorphism, and microsatellite analyses. In these applications, the reliability of the amplification techniques is particularly important. In PCR-based approaches, the plateau effect can seriously alter the original relative copy number of certain chromosomal regions. To eliminate this distorting effect, we improved the standard degenerate oligonucleotide-primed PCR (DOP-PCR) technique by following the amplification status with quantitative real-time PCR (QRT-PCR). With real-time detection of the products, we could eliminate DNA overamplification. Probes were prepared from 10 different tumor samples: primary and metastatic melanoma tissues, epidermoid and bronchioloalveolar lung carcinomas, 2 renal cell carcinomas, 2 colorectal carcinomas, and a Conn and Cushing adenoma. Probes were generated by using nonamplified and amplified genomic DNA with DOP-PCR and DOP-PCR combined with QRT-PCR. To demonstrate the reliability of the QRT-PCR based amplification protocol, altogether 152 relative copy number changes of 44 regions were determined. There was 85.6% concordance in copy number alterations between the QRT-PCR protocol and the nonamplified samples, whereas this value was only 63.8% for the traditional DOP-PCR. Our results demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA than standard DOP-PCR techniques more accurately.     

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.     

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.     

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.     

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.     

Sequential application of interphase-FISH and CGH to single cells

A comprehensive genomic analysis of single cells is needed for numerous scenarios in tumor genetics, clinical diagnostics and forensic application. PCR protocols were developed which allow an unbiased amplification of the whole genome of a single cell for subsequent analyses by comparative genomic hybridization (CGH). However, verification of single-cell CGH results has been impossible as the procedure naturally involves the destruction of the respective cell. Here we show that the genome of individual cells can be analyzed by two different single cell techniques applied sequentially to the same cell. In a first step, interphase fluorescence in situ hybridization (FISH) is applied. After evaluation of the interphase-FISH signals, cells of interest can be selected for a further analysis. Single cells are collected by laser microdissection, the DNA is amplified by linker-adaptor PCR and subjected to CGH-analysis. This strategy offers new opportunities for a sophisticated selection of cells based on interphase-FISH signals. Furthermore, the sequential application of two different single-cell approaches to the same single-cell represents the only option to control and verify the single-cell CGH results. We demonstrate the feasibility of this approach with a series of experiments including cells from pre- and postnatal diagnostics, for example, cells with trisomies 13, 18, or 21, respectively, leukemia and tumor cells and tissue sections.     

Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization

Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.     

Detection of amplified oncogenes by genome DNA microarrays in human primary esophageal squamous cell carcinoma: comparison with conventional comparative genomic hybridization analysis

Oncogene amplification in 20 surgically resected esophageal squamous cell carcinomas (ESCC) was examined with DNA microarrays that detected 57 oncogenes and two reference DNA. Alterations in DNA copy numbers detected by microarrays were compared to those obtained by conventional comparative genomic hybridization (CGH). Amplification of eight oncogenes (CCND1, FGF3/FGF4, EMS1, SAS, ERBB2, PDGFRA, MYC, and BCL2) was detected by DNA microarrays in 9 of 20 tumors. Although ERBB2 was 23.2 times higher than the control level in one case, the average magnitude of gene amplification was approximately two to four times that of the control level. EMS1, CCND1, and FGF3/FGF4, which are all located on 11q13, were amplified in 7, 5, and 4 of 20 ESCC, respectively, and they were coamplified in 3 tumors. A comparison of genome DNA microarrays and CGH data revealed that although most amplified oncogenes were included in chromosomal regions for which DNA copy number gains were detected by conventional CGH, not all amplified genes on microarrays showed concomitant DNA copy number gains on CGH. In conclusion, microarrays of oncogenes are useful for the comprehensive identification of amplified oncogenes and for analysis of areas of specific amplification within chromosomal regions with DNA copy number increases detected by CGH analysis.     

Individual identification by DNA polymorphism using formalin-fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin-fixed material is very limited. In the present study the use of 6 week formalin-fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin-fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin-fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide-primed PCR (DOP-PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP-PCR products. Furthermore, large molecular size fragments were purified from the DOP-PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.     

Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.