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.     

Characterisation and confirmation of rare beta-thalassaemia mutations in the Malay, Chinese and Indian ethnic groups in Malaysia

AIMS: In Malaysia, about 4.5% of the Malay and Chinese populations are heterozygous carriers of beta-thalassaemia. The initial identification of rare beta-globin gene mutations by genomic sequencing will allow the development of simpler and cost-effective PCR-based techniques to complement the existing amplification refractory mutation system (ARMS) and gap-PCR used for the identification of beta-thalassaemia mutations. METHODS: DNA from 173 beta-thalassaemia carriers and five beta-thalassaemia major patients from the Malay, Chinese and Indian ethnic groups were first analysed by ARMS and gap-PCR. Ninety-five per cent (174/183) of the 183 beta-globin genes studied were characterised using these two techiques. The remaining nine uncharacterised beta-globin genes (4.9%) were analysed using genomic sequencing of a 904 bp amplified PCR product consisting of the promoter region, exon 1, intervening sequence (IVS) 1, exon 2 and the 5' IVS2 regions of the beta-globin gene. RESULTS: The rare beta-globin mutations detected in the Chinese patients were CD27/28 (+C) and CD43 (GAG-TAG), and -88 (C-T) in an Indian patient. Beta-globin mutations at CD16 (-C), IVS1-1 (G-A), IVS2-1 (G-A), -86 (C-G) and Haemoglobin South Florida (CD1, GTG-ATG) were confirmed in the Malay patients. CONCLUSIONS: The seven rare beta-globin mutations and a rare haemoglobin variant confirmed in this study have been described in other populations but have not been previously described in Malaysian beta-thalassemia patients.     

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.     

限制酶切指纹-单链构象多态性--一种有效的大片段中突变的检测方法

目的；建立一种简便、价廉、有效地在大片段中检测突变的限制酶切指纹-单链构象多态性（restriction endonuclease fingerprinting-single strand conformation polymorphis，REF-SSCP）方法。方法：以耳聋患者基因组DNA为模板，扩增Cx26基因片段，通过对扩增产物进行单链构象多态性、限制酶切指纹-单链构象多态性和DNA测序，比较REF-SSCP技术对大片段中突变的检出效率。结果：长度为724bp的扩增产物SSCP结果未显示差异，REF-SSCP显示3种不同带型，经测序发现该基因中的79G→A突变，突变情况与REF-SSCP带型完全吻合，检出率为100%。结论：所建立的REF-SSCP技术适用于大样本量地检测大片段DNA中的突变。     

Use of multiple displacement amplification in the investigation of human papillomavirus physical status

BACKGROUND AND AIMS: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities. METHODS: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation. RESULTS: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples. CONCLUSIONS: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.     

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.     

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393–402, 1998. © 1998 Wiley-Liss, Inc.     

Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.     

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.     

Simultaneous sequencing of multiple polymerase chain reaction products and combined polymerase chain reaction with cycle sequencing in single reactions

DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical.     

Direct resequencing of the complete ERBB2 coding sequence reveals an absence of activating mutations in ERBB2 amplified breast cancer

Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes.     

High-Resolution Melting Analysis as a Sensitive Prescreening Diagnostic Tool to Detect KRAS , BRAF , PIK3CA , and AKT1 Mutations in Formalin-Fixed, Paraffin-Embedded Tissues

Context .-As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach. Objective .-To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations. Design .-High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing. Results .-We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays. Conclusions .-High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.     

Genetic screening for alpha-thalassemia deletional determinants by GapPCR method

The majority of a-thalassemia results from the large deletions in a-globin gene cluster, including both or either one of alpha-globin genes (alpha1 and alpha2). Most common a-thalassemia-2 deletions (single gene deletions) are -alpha3.7 and -alpha4.2, and alpha-thalassemia-1 deletions (double gene deletions) are --SEA, --THAI, --FIL, --MED and -(alpha)20.5 Although it is not easy to diagnose these deletions because of the high GC content at this locus and the sequence homology among psi alpha2, psi alpha1, alpha2 and alpha1 genes, these alleles can now be diagnosed by a single tube multiplex gapPCR assay. We showed here two Saudi Arabian patients with a-thalassemia trait who could be determined their gene mutations according to the method of Chong SS et al. (2000). [Case 1: 21-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, whereas PCR of both a-globin genes showed no amplifications. The results indicate case 1 is a homozygote of -alpha3.7(-alpha3.7/-alpha3.7). [Case 2: 31-year-old male] GapPCR assay revealed the amplification of only -alpha3.7, and PCR of both alpha globin genes showed normal amplification. DNA sequencing of the amplified a-globin genes revealed a point mutation in the poly A site of alpha2-globin gene (AATAAA-->AATAAG), which is known as alpha(T-Saudi). Thus, case 2 was confirmed to be a compound heterozygote of -alpha3.7 and alpha(T-Saudia) alpha(-alpha3.7 / alpha(T-Saudi) alpha). This gapPCR assay is a rapid, reliable screening test for common alpha-thalassemia deletions and seems to be useful for the diagnosis of thalassemic patients without an increase of Hb A2 and/or an abnormality of beta-globin gene.     

三种测序DNA模板制备方法的比较○

背景：DNA模板质量对DNA序列测定起着至关重要的作用。 目的：为基因组DNA或甲基化DNA测序寻找一种经济,简便的方法。 方法：分别采用96管集合板及96孔板提取质粒,并且针对质粒设计一对包含目的片段的引物,扩增后纯化PCR产物,通过以上3种方法制备DNA测序模板进行测序。 结果与结论：实验所采用的3种方法对于基因组DNA测序效果无差异(P > 0.05)。对于甲基化DNA测序效果,96管集合板法优于其他2种方法(P < 0.05)。说明3种方法均适用于基因组DNA的测序,而96管集合板法更适用于甲基化DNA的测序。      

单细胞水平检测von Hippel-Lindau病基因突变的实验研究

目的建立单细胞水平检测vonHippel-Lindau病基因(VHL)突变的实验方法。方法单个淋巴细胞基于多重置换扩增(multiple displacement amplification,MDA)的全基因组扩增后进行常规PCR后测序和实时荧光定量PCR,结合各荧光的终点变化判断对应的等位基因存在与否。结果MDA后单细胞扩增效率为90.91%,污染率为0;通过测序,患者VHL等位基因的脱扣率为26.67%,诊断正确率为73.33%;通过结合相应荧光的终点变化判断患者VHL基因的等位基因的脱扣率为16.67%,正确率为83.33%。结论单细胞MDA后常规PCR后测序及实时荧光定量PCR能够特异、准确地检测单个淋巴细胞的VHL基因型,两者的联合应用有助于提高检测的准确性。     

Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR

Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the "gold standard" for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.     

家族性腺瘤样息肉病中APC基因的胚系突变分析

目的 探索有效的突变检测技术，系统分析家族性腺瘤样息肉病(familial adenomatous polyposis，FAP)相关基因结肠腺瘤病(adenomatous polyposis coli，APC)基因的胚系突变，及其与疾病表型的关系。方法 从22例临床确诊的FAP患者，外周静脉血中提取基因组DNA。变性高效液相色谱、蛋白截短检测、测序技术结合应用进行全基因分析。根据患者临床资料，进行基因型-表型分析。结果 22例FAP患者中13例检出APC基因胚系突变，均为无义或移码突变。基因型-表型关系的初步分析表明，在基因5′端或3′端发生突变的患者临床症状较轻，在基因中段发生突变的患者临床症状典型或严重。结论 本研究中所采用的技术体系可敏感、高效地检出APC基因突变，APC基因的突变型与FAP患者的临床表型存在关联，所采用的技术体系适用于FAP症状出现前的基因诊断。     

Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: analysis of potential premature termination codons

Stickler syndrome is one of the milder phenotypes resulting from mutations in the gene that encodes type-II collagen, COL2A1. All COL2A1 mutations known to cause Stickler syndrome result in the formation of a premature termination codon within the type-II collagen gene. COL2A1 has 10 in-frame CGA codons, which can mutate to TGA STOP codons via a methylation-deamination mechanism. We have analyzed these sites in genomic DNA from a panel of 40 Stickler syndrome patients to test the hypothesis that mutations that cause Stickler syndrome preferentially occur at these bases. Polymerase chain reaction (PCR) amplification of genomic DNA containing each of the in-frame CGA codons was done by one of two methods: either using primers that amplify DNA that includes the CGA codon, or using allele-specific primers that either amplify normal sequence containing a CGA codon or amplify a mutant sequence containing a TGA codon. Analysis of PCR products by restriction endonuclease digestion or sequencing demonstrated the presence of a normal or mutated codon. TGA mutations were identified in eight patients, at five of the 10 in-frame CGA codons. The identification of these mutations in eight of 40 patients demonstrates that these sites are common sites for mutations in individuals with Stickler syndrome and, we propose, should be analyzed as a first step in the search for mutations that result in this disorder.     

两个中国常染色体显性遗传性多囊肾病家系的表型与基因型分析

目的研究中国人多囊肾病基因1（polycystic kidney disease 1 gene，PKD1）突变的特点，检测基因突变位点。方法25例多囊肾患者，正常对照16名，扩增PKD1基因的第44、45外显子的基因片段，变性梯度凝胶电泳突变检测系统进行初筛，然后测序。结果发现1个移码突变（12431delCT）、1个无义突变（C12217T）、1个多态性（A50747C），突变检测率为8％（2／25）。结论检测到2个新的可能的致病突变：1个移码突变（12431delCT）、1个无义突变（C12217T）。