高保真酶特异性检测大鼠SNP基因型新方法

目的应用高保真酶（Pfu）和3’末端修饰引物在单管双向等位基因特异性扩增（SB-ASA）中区分SNP基因型,建立高保真酶特异性检测SNP基因型的新方法。方法选取近交系大鼠SNP位点,以RS8149053为例,设计两个外部引物和两个等位基因特异性引物,四引物3’末端进行硫代磷酸化修饰,应用高保真聚合酶（Pfu）进行特异性扩增,扩增结果测序验证其可靠性。结果在RS8149053 SNP位点（C/T）上,等位基因型CC扩增出179 bp目的片段,基因型TT扩增出597 bp目的片段,基因型不同则扩增出分子量不同的片段,目的条带测序结果与Rat Genome Database数据库基因型结果一致,高保真酶扩增结果稳定且特异性强。结论高保真酶等位基因特异性扩增技术能有效降低假阳性率,是一种快速、特异的SNP基因分型新方法。     

Genotyping on ALDH2: Comparison of Four Different Technologies

Objectives

This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and Methods

We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results

All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions

All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.     

An innovative SNP genotyping method adapting to multiple platforms and throughputs

Key message

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility.

Abstract

Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3′ regions to significantly increase the amplification specificity of the two alleles and tailed at 5′ ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion–deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis

Isolated-probe PCR (IP-PCR) is a method that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting while maintaining a closed tube system. A double-stranded DNA (dsDNA) dye LCGreen I was used to detect the unlabeled probes. LCGreen I is also used to detect the 277-base pair PCR product peak as an internal amplification control. To accomplish this, IP-PCR separates the asymmetric PCR amplification step and the detection step of the unlabeled probes. This prevents the probes from interfering with the amplification of the DNA target. The samples are then melted using a high-resolution DNA melting instrument: the HR-1. The closed tube system virtually eliminates PCR product contamination or sample carryover The target apolipoprotein E (APOE) was chosen to test the IP-PCR technique. APOE contains two single nucleotide polymorphisms (SNPs) located 139 base pairs apart in a GC-rich region of the human genome. The results from this study show that the IP-PCR technique was able to determine the correct APOE genotype for each of the 101 samples. The IP-PCR technique should also be useful in detecting SNPs in other high-GC regions of the human genome.     

Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons

Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

High resolution melting analysis for detection of variable number of tandem repeats polymorphism of XRCC5

Several polymorphisms in the XRCC5 (X-ray repair cross-complementing 5; OMIM: 194364) were reported. Polymorphism of variable number of tandem repeats (VNTR) in the promoter region of XRCC5 (rs6147172) was reported. The main aim of the present study is to introduce the high resolution melting analysis (HRMA) method for genotyping of the polymorphism of XRCC5 VNTR. Genotypes of XRCC5 VNTR were determined by HRMA and conventional PCR method, and confirmed by DNA sequencing. The results for genotyping using HRMA and conventional PCR showed 100% concordance. All genotypes of the XRCC5 VNTR polymorphism could be accurately detected by HRMA.     

Apolipoprotein E genotyping by multiplex tetra-primer amplification refractory mutation system PCR in single reaction tube

Apolipoprotein E (APOE) plays a critical role in lipoprotein metabolism by binding to both low-density lipoprotein and APOE receptors. The APOE gene has three allelic forms, epsilon2, epsilon3, and epsilon4, which encode different isoforms of the APOE protein. In this study, we have developed a new genotyping method for APOE. Our multiplex tetra-primer amplification refractory mutation system (multiplex T-ARMS) polymerase chain reaction (PCR) was performed in a single reaction tube with six primers consisting of two common primers and two specific primers for each of two single nucleotide polymorphism (SNP) sites. We obtained definitive electropherograms that showed three (epsilon2/epsilon2, epsilon3/epsilon3, and epsilon4/epsilon4), four (epsilon2/epsilon3 and epsilon3/epsilon4), and five (epsilon2/epsilon4) amplicons by multiplex T-ARMS PCR in a single reaction tube. Multiplex T-ARMS PCR for APOE genotyping is a simple and accurate method that requires only a single PCR reaction, without any another treatments or expensive instrumentation, to simultaneously identify two sites of single nucleotide polymorphisms.     

Determining SNP allele frequencies in DNA pools

Single nucleotide polymorphisms (SNPs) are among the most common types of polymorphism used for genetic association studies. A method to allow the accurate quantitation of their allele frequencies from DNA pools would both increase throughput and decrease costs for large-scale genotyping. However, to date, most DNA pooling studies have concentrated on the use of microsatellite polymorphisms. In the case of SNPs that are restriction fragment length polymorphisms (RFLPs), studies have tended to use methods for the quantitation of allele frequency from pools that rely on densitometric evaluation of bands on an autoradiograph. Radiation-based methods have well-known drawbacks, and we present two alternative methods for the determination of SNP allele frequencies. For RFLPs, we used agarose gel electrophoresis of digested PCR products with ethidium bromide staining combined with densitometric analysis of gel images on a PC. For all types of SNP, we used allele-specific fluorescent probes in the Taqman assay to determine the relative frequencies of two different alleles. Both methods gave accurate and reproducible results, suggesting they are suitable for use in DNA pooling experiments.     

基于四引物扩增受阻突变体系PCR 的多重乳腺癌相关SNP 位点检测方法的建立

为提高单核苷酸多态性检测的通量, 引入多重嵌合引物PCR 和毛细管电泳对四引物扩增受阻突变体系PCR 进行改进. 针对乳腺癌位点rs4784227(C>T), rs1219648(G>A)和rs3803662(T>C)设计特异性嵌合引物, 经一次PCR扩增后, 通过毛细管电泳分析产物长度, 同时确定3 个位点的基因型. 70 份全血和口腔拭子样本, 电泳结果均与测序一致, 实现成功分型. 本方法仅需一次PCR 和一次毛细管电泳即可获得3 个位点的分型结果, 操作简单、快速准确.     

Interleukin-8-251T > A, Interleukin-1α-889C > T and Apolipoprotein E polymorphisms in Alzheimer's disease

An inflammatory process has been involved in numerous neurodegenerative disorders such as Parkinson's disease, stroke and Alzheimer's disease (AD). In AD, the inflammatory response is mainly located in the vicinity of amyloid plaques. Cytokines, such as interleukin-8 (IL-8) and interleukin-1α (IL-1α), have been clearly involved in this inflammatory process. Polymorphisms of several interleukin genes have been correlated to the risk of developing AD. The present study investigated the association of AD with polymorphisms IL-8 -251T > A (rs4073) and IL-1α-889C > T (rs1800587) and the interactive effect of both, adjusted by the Apolipoprotein E genotype. 199 blood samples from patients with AD, 146 healthy elderly controls and 95 healthy young controls were obtained. DNA samples were isolated from blood cells, and the PCR-RFLP method was used for genotyping. The genotype distributions of polymorphisms IL-8, IL-1α and APOE were as expected under Hardy-Weinberg equilibrium. The allele frequencies did not differ significantly among the three groups tested. As expected, the APOE4 allele was strongly associated with AD (p < 0.001). No association of AD with either the IL-1α or the IL-8 polymorphism was observed, nor was any interactive effect between both polymorphisms. These results confirm previous studies in other populations, in which polymorphisms IL-8 -251T > A and IL-1α-889C > T were not found to be risk factors for AD.     

A multiplex single-base extension protocol for genotyping Cdx2, FokI, BsmI, ApaI, and TaqI polymorphisms of the vitamin D receptor gene

The well-described role of the vitamin D endocrine system in bone metabolism makes its receptor a widely investigated candidate gene in association studies looking for the genetic basis of complex bone-related phenotypes. Most association studies genotype five polymorphic sites along the gene using PCR-RFLP and allele-specific amplification methods, which may not be the better choice in large case/control or cross-sectional studies. In this case, genotyping SNPs in parallel and using automated allele-calling methods are important to decrease genotyping errors due to manual data handling and save sample in cases where the amount of DNA is limited. The aim of this study was to present a straightforward method based on multiplex PCR amplification followed by multiplex single-base extension as a simple way to genotype five vitamin D receptor gene polymorphisms in parallel, which may be implemented in medium- to large-scale case/control or cross-sectional studies. The results regarding method feasibility and optimization are presented by genotyping eight paternity trios and seven samples of Brazilian postmenopausal women who took part in an ongoing association study carried out by members of our group.     

Development of a high resolution melting method for genotyping of risk HLA-DQA1 and PLA2R1 alleles and ethnic distribution of these risk alleles

Recent studies have demonstrated that alleles at single nucleotide polymorphisms (SNPs) rs2187668 and rs4664308 within genes HLA-DQA1 and PLA2R1, respectively, had a significant impact on the susceptibility to idiopathic membranous nephropathy (IMN). Analysis of the two genomic loci could identify alleles for individuals at risk for IMN. Conventional methods for genotyping are labor intensive, expensive or time consuming. High resolution melting (HRM) is a new technique for genotyping and has the advantages of simplicity, speed, high sensitivity and low cost. Here, we describe genotyping of SNPs rs2187668 and rs4664308 using HRM. In this study, we identified polymorphisms of rs2187668 and rs4664308 in 480 healthy unrelated Chinese volunteers of two ethnic groups from three different geographical areas in China. The two genomic loci were genotyped by HRM using a saturating fluorescent dye SYTO® 9 on 7900 HT and RG 6000 instruments, and were further confirmed by direct DNA sequencing. Three different SNP genotypes were sufficiently distinguished by HRM with mean sensitivity of 98.8% and mean error rate of 1.9%. In addition, the allele frequencies varied greatly based on ethnic or geographic origins. In conclusion, HRM is a rapid, cost efficient, sensitive, suitable technique for genotyping, and simple enough to be readily implemented in a diagnostic laboratory. We believe this will be a valuable technique for determining the genotype of rs2187668 and rs4664308 and for assessing individual susceptibility to IMN.     

Rapid detection of the 46C --> T polymorphism in the factor XII gene, a novel genetic risk factor for thrombosis, by melting peak analysis using fluorescence hybridization probes

Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C --> T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C --> T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C --> T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C --> T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.     

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的：设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法：设计的电化学基体由印刷电路板（PCB）组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果：该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论：本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia

Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.     

Determination of allele frequency in pooled DNA: comparison of three PCR-based methods

Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).