A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.     

High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SNiPTag)

Single nucleotide polymorphisms (SNPs) are becoming widely recognized as the new currency for gene mapping as increasing numbers are discovered. Here we outline a method for their rapid analysis based on an allele-specific polymerase chain reaction (PCR) which employs a competitive approach, whereby both allele-specific primers are present in the same reaction and carry different fluorescent labels. This procedure is simple and amenable to high-throughput genotyping using conventional automated sequencing equipment, and no post-PCR modifications are required. Verification of the procedure was carried out by comparison of results derived by this method with those from restriction enzyme digestion of the ALDH2 exon 12 functional polymorphism (Glu-487-Lys) in 109 individuals. Additionally, we have examined all combinations of nucleotide substitutions and shown them to be differentiated by this method. As proof of concept, several assays were combined and loaded on a single gel lane/capillary to substantially improve throughput. This was made possible by designing the PCR products to be of different lengths and no interference was observed between products differing in size by only six nucleotides. We outline a number of test assays for well-characterized SNPs in human candidate genes for behavioral disorders.     

A simple method for genotyping the bovine growth hormone gene

An allele-specific polymerase chain reaction (PCR) amplification method was developed to determine the genotypes at the bovine growth hormone locus that result from two nucleotide substitutions in exon 5 of the gene. This method was a multiplex PCR (ASM–PCR) employing a common primer pair and two allele-specific reverse primers. The common primer pair was designed to amplify a target region containing two substitution points from the three variants of the bovine growth hormone gene. The allele-specific primers were designed to be mismatched with other genotypes at the 3' end of oligonucleotides. When the common and allele-specific reverse primers competed with each other, the shorter allele-specific fragments were amplified preferentially. Consequently, the PCR products of the variant-specific fragments were 347, 483 and 656 bp for alleles A, B and C, respectively, of the bovine growth hormone gene. Genotypes of the bovine growth hormone gene were easily identified by agarose gel electrophoresis of PCR products. The results suggested that this multiplex PCR method would be useful for identification of genetic variants caused by point mutations.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

A Unique Primer with an Inosine Chain at the 5′-Terminus Improves the Reliability of SNP Analysis Using the PCR-Amplified Product Length Polymorphism Method

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method.     

一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。     

Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction

Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.     

A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

A microarray-based method has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin- terminated allele-specific PCR products are spotted unpurified onto streptavidin-coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele- specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.     

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.     

Bioluminescent reporters for identification of gene allelic variants

A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca²⁺-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G??A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3??-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.     

Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3′ end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping.     

Specific authentication of Hippocampus based on SNP markers

Seahorses, which are used as traditional Chinese medicine, have large market demands and premium price. Because of overfishing, many seahorse species are endangered and listed in IUCN Red List. The purpose of this study was to develop a simple, rapid and reliable molecular method for specific authentication of seahorse species. Cytochrome c oxidase subunit I (COI) sequences were analyzed for molecular authentication of seahorses in this study. Many single nucleotide polymorphism (SNP) sites were detected among COI sequences of nine seahorse species. Based on the SNP sites, four pairs of modified specific primers were designed respectively for authentication of Hippocampus kuda, Hippocampus japonicus, Hippocampus trimaculatus and Hippocampus kelloggi using allele-specific PCR. Moreover, a multiplex allele-specific PCR for simultaneous authentication of H. kuda, H. japonicus and H. trimaculatus was also developed. The study showed that allele-specific PCR required no sequencing and electrophoresis, and the only necessity is to add fluorochrome into PCR products. Hence the whole authentication procedure only takes about 1.5 h. This work provides an effective method for rapid and reliable identification of seahorse species.     

Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.     

Universal method for single nucleotide substitution identification

A new approach to the identification of point mutations by allele-specific PCR was proposed. The mutation R408W of the human phenylalanine hydroxylase gene was used as a model. A high specificity of the approach was achieved by the use of primers partially complementary to the genomic DNA. Polyethylene glycol covalently attached to one of the allele-specific primers provides for the differential identification of the PCR products due to a change in electrophoretic mobility.     

Km typing with PCR: application to population screening.

The immunoglobulin kappa light chain (IgK) locus may play a significant role in the pathology of both infectious and autoimmune diseases. Most of the work on IgK genetics has been conducted using immunological techniques for allelic typing and sequence analysis. This is restricted by availability of reagents and can be both expensive and time-consuming. PCR primers were designed to amplify the kappa constant gene (Ck), and four allele-specific oligonucleotides (ASOs) were used to distinguish the alleles in the amplified PCR products. Direct sequencing of PCR products was performed to confirm that the primers specifically amplified the Ck region and the ASOs differentiated the Km alleles. Sequencing of an average of 209 nucleotides of DNA from 50 individuals revealed no variation except at codon 191, which is known to be involved in a frequent polymorphism. An analysis of 347 different individual DNAs from 10 human populations was conducted to determine Km allelic frequencies within these populations and to apply this type of data collection to population studies.     

A rapid and reliable method for direct genotyping of codon 360 in the human apolipoprotein A-IV gene.

Human apolipoprotein A-IV exhibits a polymorphism, first investigated at the protein level, that is caused by a single amino acid substitution of glutamine to histidine at codon 360. Detection of this polymorphism requires polymerase chain reaction (PCR) and direct sequencing of the amplified products, radiolabeled allele-specific oligonucleotides (ASOs) technique, or restriction enzyme analysis of the amplified products. However, these techniques involve the use of radioactivity and/or are not well suited to the rapid processing of a large number of samples. In this paper, we propose a new technique, a bispecific-allele primer amplification, in which a simple electrophoresis of PCR products is used for typing the variation at codon 360. The 3' primer of PCR hybridizes with one or other homologous sequence in the apoA-IV gene, depending on the presence or the absence of the mutation. This differential hybridization of the primer is used for typing the variation. In order to demonstrate the validity of this system, 120 individuals phenotyped by two-dimensional electrophoresis and genotyped by ASO were analyzed by this new technique. The results obtained by the latter method are in agreement with those found by the other techniques. However, this method is simple, more reliable, and will facilitate population studies without using radioactive materials.     

Rapid and efficient detection of genetic polymorphism in wheat through amplification by polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify genomic DNA of several wheat genotypes. The oligonucleotides used as primers were the terminal sequences of a gamma-gliadin gene. The electrophoretic analysis of the PCR products showed specific bands which revealed both inter- and intra-specific genetic polymorphism among the examined genotypes. The technique is proposed as a very simple and efficient alternative to RFLP markers.     

一种基于适配器连接介导的等位基因特异性扩增法测定多重SNP

建立了一种基于DNA适配器连接介导的等位基因特异性扩增法测定多重SNP。以CYP2D6基因中的5个SNP位点（100C>T,1661G>C,1758G>T,2470T>C和2850C>T）为例,用PCR法预扩增得一段含所有待测SNP位点的长片段,然后用限制性内切酶将其消化成短片段,在连接酶的作用下与设计的DNA适配器（adapter）相连;该适配器的一端与限制性内切酶降解后留下的粘性末端相同,另一端带有一段公共序列。在两管中加入与适配器连接的片段作为PCR扩增模板,并分别加入SNP特异性引物和一种适配器特异性的通用引物进行PCR扩增,最后用凝胶电泳法分离PCR扩增产物。由于每管与SNP的两种特异性引物中的一种对应,可以根据每管中扩增片段的大小判断SNP的类型。通过凝胶电泳法可以一次分离与5种SNP类型相对应的引物特异性延伸反应产物;采用该法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与限制性片段长度多态性法（RFLP）测定结果完全一致。该方法采用n+1种引物（n种SNP特异性引物和一种通用引物）进行n重PCR反应,极大提高了PCR反应的特异性,结果准确,可用于同时测定多个SNP位点。