Typing single-nucleotide polymorphisms using a gel-based sequencer

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot™ kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotype (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.     

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.     

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks.In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

Multiplex SNP-SCALE: a cost-effective medium-throughput single nucleotide polymorphism genotyping method

We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-μL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.     

中德生物STR荧光ZDgenetype 9+1基因分型试剂盒应用结果分析

目的:确证中德生物STR荧光ZDgenetype 9 1基因分型试剂盒应用于个体基因分型的准确性和重复性。方法:应用中德生物ZDgenetype 9 1 STR荧光基因分型试剂盒扩增100例无关个体样本,建立扩增产物在ABI 310遗传分析仪上基因分型的方法,相同样本扩增结果与AmpFLSTR Identifiler基因分型结果进行比较。结果:ZDgenetype 9 1荧光基因分型试剂盒对100例无关个体血样进行复合扩增并实现了在ABI 310遗传分析仪上进行基因分型,其结果与AmpFLSTR Identifile扩增相同样本的基因分型结果吻合率为100%。结论:中德生物ZDgenetype 9 1 STR荧光基因分型试剂盒适用于法庭科学日常办案和科研使用。     

Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy.

We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.     

Human telomeric DNA sequences are a major target for the antitumour drug bleomycin

The DNA sequence specificity of the cancer chemotherapeutic agent bleomycin was examined in a human telomeric DNA sequence and compared with that of non-telomeric sequences. The target DNA sequence contained 17 repeats of the human telomeric sequence and other primary sites of bleomycin cleavage. The 377-base-pair target DNA was fluorescently labelled at the 3′-end, damaged with bleomycin and electrophoresed in an ABI 3730 automated capillary sequencer to determine the intensity and sequence specificity of bleomycin damage. The results revealed that bleomycin cleaved primarily at 5′-GT in the telomeric sequence 5′-GGGTTA. Maxam–Gilbert chemical sequencing reactions were utilised as DNA size markers to determine the precise sites of bleomycin cleavage. The telomeric region contained strong sites of bleomycin cleavage and constituted 57% of the 30 most intense bleomycin damage sites in the DNA sequence examined. These data indicated that telomeric DNA sequences are a major site for bleomycin damage.     

猪微卫星标记多重PCR扩增组合

采用多重PCR的方法,以快速扩增微卫星标记和节约试剂为目标,对其反应条件进行优化后,获得了46个扩增效果理想的微卫星标记多重PCR组合,其中30个为二重PCR,16个为三重PCR。实验结果表明,这些多重PCP反应的引物浓度为0.06~0.3μmol/L, Mg2+的浓度变化范围为1.5~3.0mmol/L,采用的PCR缓冲液的倍数为1.0、1.2、1.4或1.6,每个PCR反应聚合酶的用量在0.2和0.4U之间,退火温度及反应循环数分别为52~60℃ 和32~50℃。所有多重PCR进一步合并为17个可在ABI 337测序仪上进行电泳的组合。 Abstract:In order to rapidly amplify pig microsatellite markers and save materials,multiplex PCR was used and its reaction condition was optimized.Forty-six combinations of multiplex PCR with good effects were obtained.Thirty of them are duplex-PCRs,sixteen are triplex-PCRs.The results of multiplexes showed that the concentration of primers varied among 0.06~0.3μmol/L,the Mg2+ concentration among 1.5~3.0 mmol/L;0.2~0.4 U of Taq polymerase and 1.0-,1.2-,1.4-,1.6-fold buffer were used,the annealing temperature and the cycle number varied among 52~60℃ and 32~50℃,respectively.All multiplexes were further combined into 17 sets for the electrophoresis on ABI 377 sequencer.     

Evidence for association of D1S249 locus on human chromosome 1 with the susceptibility to essential hypertension in Han Chinese

Essential hypertension (EH) is thought to result from the interaction of environmental and genetic factors. The molecular genetics of EH has witnessed considerable progress during the past few years. However, the number of genes involved, their chromosomal location and the magnitude of their effect on EH susceptibility are unknown. We conducted the present study to screen susceptibility genes to essential hypertension using a genome-wide scanning method in a group of Han people from Fangshan district located in the southwest of Beijing. A case-control study and affected sibpair were performed. Genotyping was carried out using a fluorescence-based semiautomated technique on automated DNA sequencer (ABI 377, PE). The basis for the genome-screen was the ABI prism linkage mapping sets of 400 microsatellite markers (version 2, PE, Co.). PCR for amplification of markers was carried out as multiplex reactions with Ampli Taq gold (PE, Co.) following protocols developed in our laboratory. Data were exported as a     

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Evidence for association of D1S249 locus on human chromosome 1 with the susceptibility to essential hypertension in Han Chinese

Essential hypertension (EH) is thought to result from theinteraction of environmental and genetic factors. The molecular genetics of EH has witnessed considerable progress during the past few years. However, the number of genes involved, their chromosomal location and the magnitude of their effect on EH susceptibility are unknown. We conducted the present study to screen susceptibility genes to essential hypertension using a genome-wide scanning method in a group of Han people from Fangshan district located in the southwest of Beijing. A case-control study and affected sibpair were performed. Genotyping was carried out using a fluorescence-based semiautomated technique on automated DNA sequencer (ABI 377, PE). The basis for the genome-screen was the ABI prism linkage mapping sets of 400 microsatellite markers (version 2, PE, Co.). PCR for amplification of markers was carried out as multiplex reactions with Ampli Taq gold (PE, Co.) following protocols developed in our laboratory. Data were exported as a text file from genotyper for subsequent two-point affected sibpair linkage analysis. The data from case-control association study showed a linkage disequilibrium between EH and marker D1S249 locus (X2 = 14.6, P = 0.002). There are 12 alleles in the D1S249 locus. The frequency of A9 allele in hypertension was higher than in normotensives, (13.6% v.s. 2.7%, X2 = 6.30, p = 0.01, OR = 4.57, 95%CI = 1.24-25.4). The data from two-point affected sibpair linkage analysis demonstrated a linkage between EH and A9 allele, P＜0.05. It suggested that microsatellite marker D1S249 locus might be associated with the genetic susceptibility to essential hypertension in Han Chinese.     

A multiplexing single nucleotide polymorphism typing method based on restriction-enzyme-mediated single-base extension and capillary electrophoresis

Millions of single nucleotide polymorphisms (SNPs) have been identified in recent years. This provides a great opportunity for large-scale association and population studies. However, many high-throughput SNP typing techniques require expensive and dedicated instruments, which render them out of reach for many laboratories. To meet the need of these laboratories, we here report a method that uses widely available DNA sequencer for SNP typing. This method uses a type II restriction enzyme to create extendable ends at target polymorphic sites and uses single-base extension (SBE) to discriminate alleles. In this design, a restriction site is engineered in one of the two polymerase chain reaction (PCR) primers so that the restriction endonuclease cuts immediately upstream of the targeted SNP site. The digestion of the PCR products generates a 5'-overhang structure at the targeted polymorphic site. This 5'-overhang structure then serves as a template for SBE reaction to generate allele-specific products using fluorescent dye-terminator nucleotides. Following the SBE, the allele-specific products with different sizes can be resolved by DNA sequencers. Through primer design, we can create a series of PCR products that vary in size and contain only one restriction enzyme recognition site. This allows us to load many PCR products in a single capillary/lane. This method, restriction-enzyme-mediated single-base extension, is demonstrated by typing multiple SNPs simultaneously for 44 DNA samples. By multiplexing PCR and pooling multiplexed reactions together, this method has the potential to score 50-100 SNPs/capillary/run if the sizes of PCR products are arranged at every 5-10 bases from 100 to 600 base range.     

葡萄感霜霉病基因RAPD标记的序列分析

利用Ｗｉｚａｒｄ ＤＮＡ ｃｌｅａｎ－ｕｐ ｓｙｓｔｅｍ纯化葡萄感霜霉病基因ＲＡＰＤ遗传标记的ＤＮＡ片段,用细菌质粒ｐＧＥＭ Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该片段,采用自动荧光ＤＮＡ测序仪对片段的核苷酸组成进行双向测序。来自欧洲葡萄粉红玫瑰的葡萄感霜霉病基因ＲＡＰＤ标记由８３５对核苷酸及其特定序列组成。所获的感霜霉病基因ＲＡＰＤ标记可以作为合成探针的基础,用于葡萄抗病育种过程中的早期选择及品种对霜     

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.     

SNP-minisequencing as an excellent tool for analysing degraded DNA recovered from archival tissues

SNP-minisequencing has become common in forensic genetics, especially for analysing degraded or low copy number DNA (LCN DNA). The aim of this study was to examine the usefulness of five SNP (single nucleotide polymorphism) markers for analyzing degraded and LCN DNA recovered from archival samples. DNA extractions of eight formalin-fixed paraffin-embedded (FFPE) tissues were performed and DNA fragments were amplified in one multiplex PCR (polymerase chain reaction). SNPs were identified in a minisequencing reaction and a gel electrophoresis in ABI Prism 377 Sequencer. The research confirmed the usefulness of SNP-minisequencing for analysing FFPE tissues.     

Pedigree analysis and haplotype sharing within diverse groups of Zea mays L. inbreds

The objectives of this study were the examination of genetic similarities in a diverse group of maize inbreds and an investigation of the incidence of shared haplotypes within closely related groups. Size polymorphisms from 218 mapped simple-sequence repeats (SSR) for 57 entries were detected with the ABI377 Prism system and scored with Genotyper software. The standard error for the estimated size of identical PCR products was 0.13 base pairs. Size estimates were used to examine genetic relationships among the Iodent, flint, corn belt dent, sweet corn and popcorn groups in Zea mays L. inbreds. Cluster analysis of SSR distance data from 57 entries showed similarity between the European flints (F2, F7 and EP1), CO109 and the su1 sweet corns developed in the United States. The inbred F64 from Argentina was distinct from all other entries. Close examination of two sources of B37 revealed that the Purdue University version of B37 contains a set of alleles characteristic of B73. Five groups (Iodents, European flints, the B73 group of corn belt dents, su1 sweet corn, popcorn) show persistent within-group haplotypes. Received: 17 July 2000 / Accepted: 5 January 2001