双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

PCR-RFLP genotyping assay for the Bcl I polymorphism of the beta-fibrinogen gene

Polymorphisms of the beta-fibrinogen gene have been shown to affect plasma fibrinogen levels and risk of coronary artery disease (CAD). We were interested in developing an automated, PCR-based genotyping assay for the purpose of exploring relationships between CAD and CAD-associated aortic stiffness and the Bcl I allele of the beta-fibrinogen gene. We have developed a rapid PCR-RFLP assay for the Bcl I polymorphism of the beta-fibrinogen gene. We carried out direct PCR of genomic DNA to facilitate sequencing of the flanking region of the beta-fibrinogen gene. Using this new sequence information, primers were designed which border the site of the Bcl I polymorphism. One of the primers was labeled with a fluorophore to facilitate detection of the fragments. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). We have developed an improved PCR-RFLP high-sample-throughput assay for the semiautomated detection of the Bcl I polymorphism of the beta-fibrinogen gene. This assay will support screening of large sample sizes required for population studies.     

Automated, PCR-RFLP genotyping of the urokinase gene

The urokinase-type plasminogen activator (u-PA) has been suggested to play a role in the early initiation and progression of atherosclerosis and coronary artery disease (CAD) (Grenett et aL, 1998). Recently, a common genetic polymorphism in the untranslated region of the u-PA gene was shown to be associated with syptomatic CAD. To study the possible role of this common genetic polymorphism in the u-PA gene, we have developed an automated, PCR-based assay. Automation of the PCR-RFLP genotyping of the BamHI polymorphism of the urokinase gene will support the screening of the large sample sizes required to do the population-based studies necessary to uncover disease susceptibility associations.     

应用基于适配器连接介导的等位基因特异性扩增法检测LRRK2基因的单核苷酸多态性

目的：应用基于适配器连接介导的等位基因特异性扩增法（Adapter Ligation—mediated Allele-specific Amplification,简称ALM-ASA）技术,检测与帕金森病（PD）发病相关的LRRK2基因中的4个SNP位点（6055G〉A,7153G〉A,4321C〉T和2264C〉T）,探讨该法用于筛查帕金森病相关SNP位点的可行性,研究多个SNP住点同时检测的准确性和可靠性。方法：运用ALM—ASA法原理,改进使用多重PCR法代替单一预扩增法,使用4对引物在单管中预扩增含所待测SNP位点的四段片段,通过酶切、酶连和PCR特异性扩增检测判断SNP的类型。经PCR体外定点突变实验制备的相应位点的突变阳性片段,检验方法的准确性和可靠性。结果：采用该法成功测定了20名PD病人和20名健康中国人的LRRK2基因中最受关注的4个SNP位点的多态性。并随机对其中10分样本的检测结果以测序法检测进行验证,结果完全一致。结论：ALM-ASA法极大提高了PCR反应的特异性,是一种准确、可靠,且费用低廉的SNP筛查方法,可推广应用于临床和实验室进行与PD有关的单核苷酸多态性的筛查检测。     

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.     

Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR.

Single nucleotide polymorphisms (SNPs) are the most abundant form of DNA polymorphism. These polymorphisms can be used in plants as simple genetic markers for many breeding applications, for population studies, and for germplasm fingerprinting. The great increase in the available DNA sequences in the databases has made it possible to identify SNPs by "database mining", and the single most important factor preventing their widespread use appears to be the genotyping cost. Many genotyping platforms rely on the use of sophisticated, automated equipment coupled to costly chemistry and detection systems. A simple and economical method involving a single PCR is reported here for barley SNP genotyping. Using the tetra-primer ARMS-PCR procedure, we have been able to assay unambiguously five SNPs in a set of 132 varieties of cultivated barley. The results show the reliability of this technique and its potential for use in low- to moderate-throughput situations; the association of agronomically important traits is discussed.     

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.     

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.     

Comparison of three PCR-based methods to detect a Piedmontese cattle point mutation in the Myostatin gene

Despite many recent advances in single nucleotide polymorphism (SNP) genotyping technologies, there is still the need for methodologies with a reasonable throughput. In this study, we compared three PCR-based methods for SNP detection: (1) a conventional PCR-based allele detection system with fluorescent genotyping technology, (2) a SNaPshot methodology by single nucleotide primer extension and, (3) a real-time PCR-based method by allele-specific minor groove-binder probes. These three methodologies were used to analyze 104 meat samples of a particular Italian cattle breed known for producing excellent quality meat and for a characteristic increased development of muscle mass, caused by a point mutation (C313Y) in the Myostatin gene. The analysis revealed 98 samples to be homozygous (mh/mh) and five to be heterozygous (mh/+) for the mutation whereas one sample resulted to be homozygous for the wild type (+/+). The results obtained with the three different assays were consistent. Overall, all three methodologies proved to be efficient for allelic discrimination studies; however, real-time PCR was faster and allowed to genotype up to 96 samples in a single step, minimizing the number of steps required for samples manipulation.     

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.     

Allele-specific PCR amplification due to sequence identity between a PCR primer and an amplicon : is direct sequencing so reliable?

PCR-direct sequencing (DS) is thought to be a very reliable method of determining DNA sequence and genotyping. Under certain conditions, however, DS can generate inaccurate results. Here we report a case of erroneous DS, in which a single nucleotide polymorphism (SNP) in the human PAX9 gene was mistyped due to allele-dependent PCR amplification. Examination of the amplified region showed that the 5' eight bases of one of the PCR primers were identical to the eight bases of the reverse strand downstream of the SNP, and the ninth base matched one of the alleles. Altering the primer so that it matched the other allele reversed the allele-specific inhibition. Reducing the base-pairing abolished the inhibition. Thus, the SNP was responsible for the difference in annealing efficacy of the primer and was therefore critical for the allele dependency. The allele-specific inhibition presented here can occur with any PCR primer sequence that encompasses a site that is polymorphic in the gene sequence. This phenomenon needs to be considered as a possibility when interpreting results from all PCR-based experiments. Sequence similarity between PCR primers and internal amplified regions should be considered for all methods for mutation detection and genotyping using PCR.     

A new approach to SNP genotyping with fluorescently labeled mononucleotides

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3′ end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.     

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.     

MilkProtChip--a microarray of SNPs in candidate genes associated with milk protein biosynthesis--development and validation

MilkProtChip is an oligonucleotide microarray based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for bovine milk protein biosynthesis. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5'end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxy nucleotide complementary to the template reveals the polymorphism. A total of 75 SNPs were selected among those associated directly or potentially with milk protein content. Among the 75 SNPs, 4 did not produce a positive signal. Most of the remaining SNPs produced a signal for both strands, except for 4 (one strand). In the validation step, 12 Polish Holstein bulls, 1 Polish Red bull, 1 bison (Bison bonasus), 11 Jersey cows and 25 Polish Holstein cows were screened to validate SNPs. Among the 71 selected SNPs--26 were found monoallelic, the rest showing at least two genotypes for the entire population under study. All the animals were earlier genotyped for 2-5 SNPs by PCR-RFLP and PCR sequencing and all showed complete concordance with APEX genotyping. APEX reactions showed relatively high signal frequencies: more than 0.9, 0.9-0.8 and below 0.8, for 65, 4 and 2 DNA samples, respectively. The primary application of the MilkProtChip is the simultaneous genotyping of dozens of SNPs to reveal and clarify the genetic background of milk protein biosynthesis. The chip may possibly be used for dairy cattle identification and paternity analysis, evolutionary studies, the evaluation of genetic distances between wild and domestic cattle breeds and the domestication history of bovine species.     

A PCR-RFLP test for simultaneous detection of two single-nucleotide insertions in the Connexin-26 gene promoter

Comparisons of Connexin-26 (GJB2) gene sequences available in the GenBank data base indicate the presence of a polymorphism in the promoter, but no easy method is available for the detection of this polymorphism. We have developed a PCR-RFLP test for simultaneous detection of two single nucleotide insertions (G and A) in the GJB2 promoter. The test is based on amplification of a 146-bp DNA fragment, which was digested with Mae I to detect the G insertion in the promoter. A similar digestion with Hinf I detects the A insertion. The test was validated using direct DNA sequencing of amplified DNA from 33 samples. After validation, we have used it to investigate DNA samples from 160 control subjects and 51 unrelated patients with nonsyndromic autosomal recessive deafness. All of the samples analyzed using the PCR test and DNA sequencing were found to contain both the G and A insertions in the GJB2 gene promoter. This PCR test will be useful in studying the prevalence of these two insertions in other populations.     

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Genotyping and detection of multiple infections of Toxoplasma gondii using Pyrosequencing

A Pyrosequencing assay, based on SAG2 gene polymorphisms, was designed for genotyping and detection of multiple infections of Toxoplasma gondii. The assay was tested on samples spiked with DNA from single and multiple genotypes of T. gondii and also on a DNA sample from the brain of a rat with multiple infections. To evaluate the comparative efficacy of the assay, identical samples were also analysed by PCR-restriction fragment length polymorphism (RFLP) and dideoxy sequencing. The Pyrosequencing assay was found to be superior to the two conventional techniques. Genotyping and detection of multiple alleles were possible after a single PCR assay in duplex format, from both the spiked and direct samples. The simplex PCR assay enabled accurate quantification of the different alleles in the mix. In comparison, PCR-RFLP and dideoxy sequencing were neither able to unequivocally detect multiple genotype infections, nor quantify the relative concentrations of the alleles. We conclude that Pyrosequencing offers a simple, rapid and efficient means for diagnosis and genotyping of T. gondii, as well as detection and quantification of multiple genotype infections of T. gondii.