Universal method for SNP 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.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 213–215.Original Russian Text Copyright © 2005 by Brenner, Ivanova, Pyshnyi, Morozov.     

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.     

A polymorphism in the bovine gamma-S-crystallin gene revealed by allele-specific amplification

Summary
A polymorphism was detected in the 3' untranslated region of the bovine gamma-S-crystallin gene by direct sequencing of polymerase chain reaction (PCR) products from genomic DNA of an N'Dama bull and a Boran cow. A set of three PCR primers was designed to detect this difference and thus give allele-specific amplification. The two allele-specific primers differ in length by 20 nucleotides so that the allelic products may be distinguished by simple agarose gel electrophoresis following a single PCR reaction. This provides a simple and rapid assay for this polymorphism.     

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.     

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.     

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。     

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.     

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.     

Multi-primer target PCR for rapid identification of bovine DRB3 alleles

Multi-primer target polymerase chain reaction (MPT-PCR) is a rapid method for the identification of specific BoLA-DRB3 alleles. In a single PCR reaction, the presence of two alleles associated with increased risk, DRB3.2*23 (DRB3*2701-2703, 2705-2707) and decreased risk, DRB3.2*16 (DRB3*1501, 1502), of mastitis in Canadian Holstein can be detected. Two outer primers amplify exon 2 of DRB3. Simultaneously, two inner, allele-specific primers amplify individual alleles. Initially, 40 cows previously typed by PCR-restriction fragment length polymorphism (PCR-RFLP) were genotyped using the multi-primer approach. An additional 30 cows were first genotyped by multi-primer target PCR, then by PCR-RFLP. All animals were correctly identified and there were no false positives. This technique can readily be modified to identify other BoLA alleles of interest.     

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.     

Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays.

DNA microarray technology holds significant promise for human DNA diagnostics. A number of technical approaches directed at the parallel identification of mutations or single nucleotide polymorphisms make use of polymerase-based specificity, like minisequencing or allele-specific primer elongation. These techniques, however, require separate laborious sample amplification, preparation, and purification steps, making large-scale analyses time and cost consuming. Here, we address this challenge by applying an experimental setup using simultaneous solid and liquid phase PCR on polyethyleneimine-coated glass slides, a novel microarray support allowing on-chip amplification reactions with exquisite specificity. A gene-specific oligonucleotide tiling array contains covalently attached allele-specific primers which interrogate single nucleotide positions within a genomic region of interest. During a thermal cycling reaction amplification products remain covalently bound to the solid support and can be visualized and analyzed by the incorporation of fluorescent dyes. Using the described procedure we unequivocally defined the presence of point mutations in the human tumor suppressor gene p53 directly from a natural DNA source. This semi-multiplex solid phase amplification format allowed the rapid and correct identification of 20 nucleotide positions from minute amounts of human genomic DNA. Our results suggest that this approach might constitute a vital component of future integrated DNA chip devices used in gene analysis.     

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.     

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.     

Rapid and direct detection of the most frequent Mediterranean beta-thalassemic mutations by multiplex allele-specific enzymatic amplification.

A rapid nonradioactive method for the diagnosis of the most frequent Mediterranean beta-thalassemic mutations is described based on a multiplex allele-specific polymerase chain reaction (PCR). This method allows direct detection of normal or mutated alleles on genomic DNA. We have used this approach to detect the most frequent Mediterranean mutations: IVS-1 nt 110 (G----A) and 39 nonsense (C----T). For each mutation three allele-specific oligonucleotides were used: one common upstream primer and two downstream primers differing in their terminal 3' nucleotide (one specific for the normal allele and one for the mutant allele). For each sample two PCR reactions were performed in parallel using in one case IVS-1 nt 110 and codon 39 normal primers and in the second case using the corresponding mutated primers. In both cases the different PCR fragments were visualized. After optimization these primers directed only amplification of their complementary allele. A single blind study was performed on the DNA of 18 individuals who were homozygous or heterozygous for these mutations. In comparison with a parallel investigation, using oligonucleotide probes, all the results were unambiguous. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows the screening of a population group, including heterozygotes, which is required from an epidemiological and anthropological point of view. It could be extended to the large series screening of haplotypes before targeted diagnosis of various genetic diseases.     

Frequency of the 735G → A mutation of the 5′-splice donor site of intron 14 of the dihydropyrimidine dehydrogenase gene (<Emphasis Type="Italic">DPYD</Emphasis>) in residents of novosibirsk region (Russia) as revealed with fluorescent oligonucleotides

A simple method was developed for end-point fluorescence detection of the 735G → A mutation of the 5′-splice donor site of intron 14 of the dihidropyrimidine dehydrogenase gene (DPYD). The method was based on allele-specific PCR with duplex Scorpion primers. The genotyping results obtained by the fluorescent end-point PCR technique completely coincided with the results obtained by allele-specific PCR with amplicon detection in agarose gel. Genotyping was performed in 291 DNA samples from residents of Novosibirsk region (Russia), and two heterozygotes (0.69%) were detected.     

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。

     

Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli.

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

A new and versatile method for the successful conversion of AFLP markers into simple single locus markers

Genetic markers can efficiently be obtained by using amplified fragment length polymorphism (AFLP) fingerprinting because no prior information on DNA sequence is required. However, the conversion of AFLP markers from complex fingerprints into simple single locus assays is perceived as problematic because DNA sequence information is required for the design of new locus-specific PCR primers. In addition, single locus polymorphism (SNP) information is required to design an allele-specific assay. This paper describes a new and versatile method for the conversion of AFLP markers into simple assays. The protocol presented in this paper offers solutions for frequently occurring pitfalls and describes a procedure for the identification of the SNP responsible for the AFLP. By following this approach, a high success rate for the conversion of AFLP markers into locus-specific markers was obtained.     

Refinement of single-nucleotide polymorphism genotyping methods on human genomic DNA: amplifluor allele-specific polymerase chain reaction versus ligation detection reaction-TaqMan

Single-nucleotide polymorphisms (SNPs) have proven to be powerful genetic markers for a variety of genetic applications, e.g., association studies leading to dissection of both monogenetic and complex diseases. However, no single SNP genotyping method has been broadly accepted. In the present study, we compared and refined two promising methods with potential for research and for diagnostic SNP genotyping: Amplifluor allele-specific polymerase chain reaction (PCR) and ligation detection reaction (LDR)-TaqMan. The methods are based on allele-specific primer extension and allele-specific ligation, respectively. Since LDR-TaqMan had previously been tested on just Arabidopsis thaliana, we adjusted the method for the more complex human genome. Amplifluor allele-specific PCR has a single-step and closed-tube format, whereas the LDR-TaqMan assay comprises two simple steps. Contrary to the primer-extension-based method, the ligation-based method can be multiplexed. Refining the LDR-TaqMan technique, we successfully replaced a previously suggested three-step multiplexing procedure with a less laborious two-step approach. Comparing refined LDR-TaqMan with Amplifluor allele-specific PCR in a family-based study, both techniques appeared similar with respect to high robustness and accuracy. As both approaches utilize primers with common tails, all SNPs can be assayed with the same couple of fluorescence reporting reagents, ensuring low establishing and running expenses.