首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 0 毫秒
1.
High-resolution melting of polymerase chain reaction (PCR) products can detect heterozygous mutations and most homozygous mutations without electrophoretic or chromatographic separations. However, some homozygous single nucleotide polymorphism (SNPs) have melting curves identical to that of the wild-type, as predicted by nearest neighbor thermodynamic models. In these cases, if DNA of a known reference genotype is added to each unknown before PCR, quantitative heteroduplex analysis can differentiate heterozygous, homozygous, and wild-type genotypes if the fraction of reference DNA is chosen carefully. Theoretical calculations suggest that melting curve separation is proportional to heteroduplex content difference and that the addition of reference homozygous DNA at one seventh of total DNA results in the best discrimination between the three genotypes of biallelic SNPs. This theory was verified experimentally by quantitative analysis of both high-resolution melting and temperature-gradient capillary electrophoresis data. Reference genotype proportions other than one seventh of total DNA were suboptimal and failed to distinguish some genotypes. Optimal mixing before PCR followed by high-resolution melting analysis permits genotyping of all SNPs with a single closed-tube analysis.  相似文献   

2.
汪维鹏  倪坤仪  周国华 《遗传》2006,28(2):219-225
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。

  相似文献   

3.
Advances in technologies for identifying genetic polymorphisms rapidly and accurately will dramatically accelerate the discovery of disease-related genes. Among a variety of newly described methods for rapid typing of single-nucleotide polymorphisms (SNPs), gene detection using DNA microarrays is gradually achieving widespread use. This method involves the use of short (11- to 13-mer) allele-specific oligonucleotides. This method allows simultaneous analysis of many SNPs in DNAs from a large number of individuals, in a single experiment. In this work, we evaluated the accuracy of a new microarray-based short allele-specific oligonucleotide (ASO) hybridization method. There is a 96-well formatted array on a single plate, in which up to 256 spots are included in each well. Fluorescent probes for our experiments were produced by multiplex PCR amplification often target SNP-containing regions. We genotyped 192 individuals across a panel of ten single base variations, which included an insertion/deletion polymorphism. For comparison, we genotyped the same individuals for the same SNPs by the method of single-base extension with fluorescence detection. The typing accuracies of the microarray-based PCR-ASO and single-base extension methods were calculated as 99.9% and 99.1%, respectively, on the basis of genotyping results determined by direct sequencing. We conclude that the microarray-based hybridization method using short ASO probes represents a potential breakthrough technology for typing large numbers of SNPs rapidly and efficiently.  相似文献   

4.
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.  相似文献   

5.
When we placed an ENA residue into primers at the 3' end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer-dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS-PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.  相似文献   

6.
BARD1 Val507Met (1592A>G) is an interesting marker for association studies on cancer risk. However, studies are scarce in the literature, probably reflecting the methodological problem imposed by the fact that next to the 1592A>G stands the 1591C>T single nucleotide polymorphism (SNP). We have designed an allele-specific PCR method capable of molecular haplotyping tandem SNPs. In the tandem SNPs haplotyping assay (tSNPh), four reverse primers are designed to be perfect matches of each potential haplotype. The forward primer is labeled with a fluorochrome. PCR products are analyzed by capillary electrophoresis. Haplotyping is performed by size calling. To ascertain the accuracy and reproducibility of the assay, we measured the level of concordance with sequencing data in 124 samples. In vitro-generated templates have been used for further testing. We developed a novel and reliable assay that permits typing two SNPs directly adjacent to each other, avoiding mutual interferences. The method is amenable to automation and high throughput. We expect that this assay will contribute to clarifying the role of BARD1 in cancer susceptibility. In addition, we suggest that tandem SNPs are potentially interesting polymorphic markers in which molecular haplotyping can be performed easily.  相似文献   

7.
Single nucleotide polymorphisms are the most common polymorphism in plant and animal genomes and, as such, are the logical choice for marker-assisted selection. However, many plants are also polyploid, and marker-assisted selection can be complicated by the presence of highly similar, but non-allelic, homoeologous sequences. Despite this, there is practical and academic demand for high-throughput genotyping in several polyploid crop species, such as allohexaploid wheat. In this paper, we present such a system, which utilizes public single nucleotide polymorphisms previously identified in both agronomically important genes and in randomly selected, mapped, expressed sequence tags developed by the wheat community. To achieve relatively high levels of multiplexing, we used non-amplified genomic DNA and padlock probe pairs, together with high annealing temperatures, to differentiate between similar sequences in the wheat genome. Our results suggest that padlock probes are capable of discriminating between homoeologous sequences and hence can be used to efficiently genotype wheat varieties.  相似文献   

8.
We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.  相似文献   

9.

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.  相似文献   

10.
In this article, we describe a genotyping approach applicable to both individual and multiplexed single nucleotide polymorphism (SNP) analysis, based on a ligation detection reaction (LDR) performed directly on genomic DNA. During the ligation, the biallelic state of the SNP locus is converted into a bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by a 5'-nuclease assay (TaqMan) with universal fluorescent probes. The LDR-TaqMan method was successfully applied for the genotyping of 30 SNP loci of Arabidopsis thaliana. The technology is cost-effective, needs no locus-specific optimization, requires minimal manipulations, and has very good potential for automation.  相似文献   

11.
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.  相似文献   

12.
BackgroundBisoprolol is an effective β1-adrenergic blocker, an inter-individual genetic variability was recorded in its response. This study aimed at investigating the association of CYP2D6*2A (rs1080985) and CYP2D6*10 (rs1065852) single-nucleotide polymorphism (SNP) with Bisoprolol response in cardiac patients attending King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia.Patients and methodsIn the study, 107 patients were enrolled. Five mL of venous blood was collected from each patient and genotyping for CYP2D6*2A and CYP2D6*10 using Vivid® CYP2D6 Green Screening Kit (Life Technologies, USA). Response to Bisoprolol was evaluated through assessment of diastolic and systolic blood pressure and by measuring Bisoprolol plasma level using triple quad mass spectrometer (TQ-MS).ResultsAll patients were found to carry homozygous wild type CYP2D6*10 (GG) and none were carrying heterozygous (GA) or mutant homozygous (AA) genotype. CYP2D6*2A allele was detected in the homozygous wild type (GG) in 70 out of 107 patients, the heterozygous (GC) in 19 patients, and the homozygous mutant (CC) in 18 patients with minor allele frequency (MAF) of 25.7%. The plasma concentrations of Bisoprolol in CC carriers were significantly lower than those in GG & CC carriers by 25%, and 51%; respectively. Higher systolic and diastolic blood pressures were also observed in CC carriers than GG and CC carriers.ConclusionThere is a possible association of CYP2D6*2A genotype with plasma concentration of bisoprolol. This could provide a helpful tool to choose the optimum dose for bisoprolol, depending on the patient’s genotyping, in order to increase effectiveness and ameliorate its toxicity.  相似文献   

13.
Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.  相似文献   

14.
High-resolution amplicon melting is a simple method for genotyping that uses only generic PCR primers and a saturating DNA dye. Multiplex amplicon genotyping has previously been reported in a single color, but two instruments were required: a carousel-based rapid cycler and a high-resolution melting instrument for capillaries. Manual transfer of capillaries between instruments and sequential melting of each capillary at 0.1°C/s seriously limited the throughput. In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting [LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT] was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0.3°C/s, 3× the prior rate. Sample volume was reduced from 20 μl to 10 μl. In a blinded study of 109 samples (436 genotypes), complete concordance with standard assays was obtained. In addition, the rare variant MTHFR 1317T>C was genotyped correctly when present. The LS-32 simplifies more complex high-resolution melting assays by reducing hands-on manipulation, total time of analysis, and reagent cost while maintaining the resolution necessary for multiplex amplicon genotyping.  相似文献   

15.
Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.  相似文献   

16.
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.  相似文献   

17.
Over the past few years, considerable progress has been made in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, largely through the investment of the human genetics community. These technologies are well adapted to diploid species. For plant breeding purposes, it is important to determine whether these genotyping methods are adapted to polyploidy, as most major crops are former or recent polyploids. To address this problem, we tested the capacity of the multiplex technology SNPlex™ with a set of 47 wheat SNPs to genotype DNAs of 1314 lines that were organized in four 384-well plates. These lines represented different taxa of tetra- and hexaploid Triticum species and their wild diploid relatives. We observed 40 markers which gave less than 20% missing data. Different methods, based on either Sanger sequencing or the MassARRAY® genotyping technology, were then used to validate the genotypes obtained by SNPlex™ for 11 markers. The concordance of the genotypes obtained by SNPlex™ with the results obtained by the different validation methods was 96%, except for one discarded marker. Furthermore, a mapping study on six markers showed the expected genetic positions previously described. To conclude, this study showed that high-throughput genotyping technologies developed for diploid species can be used successfully in polyploids, although there is a need for manual reading. For the first time in wheat species, a core of 39 SNPs is available that can serve as the basis for the development of a complete SNPlex™ set of 48 markers.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
Liu H  Li S  Wang Z  Hou P  He Q  He N 《Biotechnology journal》2007,2(4):508-511
A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号