Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER)

A new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which is capable of detecting small allele frequency differences between two DNA pools for genetic association studies other than SNP typing. The method is based on specific primer extension reactions coupled with PPi detection. As the specificity of the primer-directed extension is not enough for quantitative SNP analysis, artificial mismatched bases are introduced into the 3′-terminal regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the primer 3′-terminus. Contamination with endogenous PPi, which produces a large background signal level in SNP analysis, was removed using PPase to degrade the PPi during the sample preparation process. It is possible to accurately and quantitatively analyze SNPs using a set of primers that correspond to the wild-type and mutant DNA segments. The termini of these primers are at the mutation positions. Various types of SNPs were successfully analyzed. It was possible to very accurately determine SNPs with frequencies as low 0.02. It is very reproducible and the allele frequency difference can be determined. It is accurate enough to detect meaningful genetic differences among pooled DNA samples. The method is sensitive enough to detect 14 amol ssM13 DNA. The proposed method seems very promising in terms of realizing a cost-effective, large-scale human genetic testing system.     

Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3' exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3' phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3' exonuclease activity and the 3' phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Single-base discrimination mediated by proofreading 3′ phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3′ exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3′ phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3′ exonuclease activity and the 3′ phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

Development of multiplex DNA electronic microarrays using a universal adaptor system for detection of single nucleotide polymorphisms

The NanoChip electronic microarray is designed for the rapid detection of genetic variation in research and clinical diagnosis. We have developed a multiplex electronic microarray assay, specific for single nucleotide polymorphism (SNP) genotyping and mutation detection, using universal adaptor sequences tailed to the 5' end of PCR primers specific to each target. PCR products, amplified by primers directed to the universal adaptor sequence, are immobilized on the microarray either directly or via capture oligonucleotides complementary to the universal adaptor sequence. This simple modification results in a significant increase in fidelity with improved specificity and accuracy. In addition, the multiplexing of genetic variant detection allows increased throughput and significantly reduced cost per assay. This general schema can also be applied to other microarray and macroarray formats.     

Single-base discrimination mediated by proofreading inert allele specific Primers

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer-extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by exo(+) DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.     

四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基引入了一个人为错配碱基 ,使引物的错误延伸率显著降低 ,大大提高了SNP分析的准确性 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .     

Multiplex single nucleotide polymorphisms genotyping using solid-phase single base extension on magnetic nanoparticles

To fulfill the increasing need for large-scale genetic research, we have developed a new solid-phase single base extension (SBE) protocol on magnetic nanoparticles (MNPs) for multiplex SNP detection using adapter polymerase chain reaction (PCR) products as templates. Extension primers were covalently immobilized on the MNPs, and allele-specific extension took place along the stretch of target DNA for one-color ddNTP incorporation. The MNPs with fluorophores were spotted on a glass slide to fabricate a “bead array” to discriminate their genotypes. Eight SNP loci of three DNA samples were interrogated, and the experiment demonstrated that it is an efficient method for large-scale SNP genotyping.     

Multiplex automated primer extension analysis: simultaneous genotyping of several polymorphisms.

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is of significant scientific importance for linkage and association studies. We report here an automated fluorescent method we call multiplex automated primer extension analysis (MAPA) that can accurately genotype multiple known SNPs simultaneously. This is achieved by substantially improving a commercially available protocol (SNaPshot). This protocol relies on the extension of a primer that ends one nucleotide 5'of a given SNP with fluorescent dideoxy-NTPs (minisequencing), followed by analysis on an ABI PRisMS 377 Semi-Automated DNA Sequencer Our modification works by multiplexing the initial reaction that produces the DNA template for primer extension and/or multiplexing several primers (corresponding to several SNPs) in the same primer extension reaction. Then, we run each multiplexed reaction on a single gel lane. We demonstrate that MAPA can be used to genotype up to four SNPs simultaneously, even in compound heterozygote samples, with complete accuracy (based on concordance with sequencing results). We also show that primer design, unlike the DNA template purification method, can significantly affect genotyping accuracy, and we suggest useful guidelines for quick optimization.     

Competitive enzymatic reaction to control allele-specific extensions

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3′-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5′ end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.     

Direct transduction of allele-specific primer extension into electrical signal using genetic field effect transistor

We have developed a genetic field effect transistor (FET) for single nucleotide polymorphism (SNP) genotyping, which is based on potentiometric detection of molecular recognition on the gate insulator. Here, we report direct transduction of allele-specific primer extension on the gate surface into electrical signal using the genetic FETs. This method is based on detection of intrinsic negative charges of polynucleotide synthesized by DNA polymerase. The charge density change at the gate surface could be monitored during primer extension reaction. Moreover, three different genotypes could be successfully distinguished without any labeling for target DNA by the use of the genetic FET in combination with allele-specific primer extension. The platform based on the genetic FETs is suitable for a simple, accurate and inexpensive system for SNP genotyping in clinical diagnostics.     

DNA聚合酶高保真机理的新发现及其在SNP分析中的应用

高保真DNA聚合酶在遗传与进化等生命活动中具有十分重要的生理与病理意义。高保真聚合酶除具有广为人知的校正功能外,最近的实验进一步表明, 由不能及时校正或难于纠正的错配碱基引发的“关”闭DNA聚合反应的效应, 同样保证了DNA聚合反应终产物的纯度。高保真聚合酶这一“关”闭DNA聚合反应的能力, 促成了其与耐外切酶消化的3´末端碱基特异性引物共同构成一个SNP敏感性纳米级复合分子“开/关”,高保真聚合酶分子中相距三纳米的聚合中心和3´→5´外切酶酶解中心则既合作又独立地起到了复合分子开关中“开”和“关”的效能:对于配对的引物,则直接在该酶的聚合中心进行聚合反应,即“开”的效应;而对于3´末端错配的引物,则从该酶的聚合中心转移至3´→5´外切酶的酶解中心,由于引物修饰了的3´末端耐外切酶的特点,继而出现了一种长时间无酶解产物的酶解过程,最后因酶的聚合中心空转而“关”闭DNA聚合反应,即“关”的效应。这一新的复合分子“开/关”在很大程度上满足了后基因时代对SNP分析的要求。该SNP分子开关的应用, 使基因诊断提高到单碱基水平。同时, 利用该方法通过SNP对基因组扫描, 在单基因遗传病病因研究及法医学鉴定上具有很强的理论和实用价值。     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Primer-design for multiplexed genotyping

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5′ end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be ‘demultiplexed’. Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.