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1.
Scheen AC Pfeil BE Petri A Heidari N Nylinder S Oxelman B 《Molecular ecology resources》2012,12(1):128-135
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. 相似文献
2.
Haplotype analysis of single nucleotide polymorphisms (SNPs) is an important and rapidly growing approach for association studies. In recent years, statistical procedures to haplotype determination from genotypic information have employed in population studies. These procedures, even though some advantages for estimation of haplotype frequencies in large population samples, have limitations in the accuracy of the analysis. In this study, we have designed a reliable method for direct haplotyping of polymorphic sites using the amplification refractory mutation system (ARMS) and restriction fragment length polymorphism (RFLP) analysis techniques. We applied the method to determination of haplotypes composed of three SNPs within the paraoxonase1 gene promoter and found the approach can be used in many studies in population and in a variety of clinical settings. 相似文献
3.
Substantial differences in bias between single‐digest and double‐digest RAD‐seq libraries: A case study
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The trade‐offs of using single‐digest vs. double‐digest restriction site‐associated DNA sequencing (RAD‐seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the remaining 384 individuals were genotyped using a double‐digest RAD‐seq (ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD‐seq versus ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations. 相似文献
4.
Galit Rotman Lina Vanagaite Francis S. Collins Yosef Shiloh 《Molecular biotechnology》1995,3(2):85-92
Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci.
The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid
isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is
digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions
that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette
linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using
this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers
at any genomic region cloned in YACs. 相似文献
5.
Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. 总被引:62,自引:12,他引:62
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R C Allen H Y Zoghbi A B Moseley H M Rosenblatt J W Belmont 《American journal of human genetics》1992,51(6):1229-1239
The human androgen-receptor gene (HUMARA; GenBank) contains a highly polymorphic trinucleotide repeat in the first exon. We have found that the methylation of HpaII and HhaI sites less than 100 bp away from this polymorphic short tandem repeat (STR) correlates with X inactivation. The close proximity of the restriction-enzyme sites to the STR allows the development of a PCR assay that distinguishes between the maternal and paternal alleles and identifies their methylation status. The accuracy of this assay was tested on (a) DNA from hamster/human hybrid cell lines containing either an active or inactive human X chromosome; (b) DNA from normal males and females; and (c) DNA from females showing nonrandom patterns of X inactivation. Data obtained using this assay correlated substantially with those obtained using the PGK, HPRT, and M27 beta probes, which detect X inactivation patterns by Southern blot analysis. In order to demonstrate one application of this assay, we examined X inactivation patterns in the B lymphocytes of potential and obligate carriers of X-linked agammaglobulinemia. 相似文献
6.
四引物扩增受阻突变体系PCR(Tetra-primer amplification refractory mutation system PCR,Tetra-primer ARMS PCR)技术是一种在普通PCR基础上发展起来的单核苷酸多态性(SNP)分型技术。该项技术综合了扩增受阻突变体系(Amplification refractory mutation system,ARMS)和四引物PCR(tetra-primer PCR)技术的优点,是对等位基因特异性PCR法的改良。它具有操作简便、分型快速、费用低廉等特点,在国内外生命科学领域尤其是遗传育种领域的应用越来越广泛。本文介绍了四引物扩增受阻突变体系PCR的技术原理及优势、结果检测手段和反应体系改进方法,并在此基础上对该技术在遗传育种研究中的应用进行综述。 相似文献
7.
Brian Patrick Hansen Metzger Gregory William Gelembiuk Carol Eunmi Lee 《Molecular ecology resources》2013,13(1):135-143
While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate. 相似文献
8.
Bundock PC Cross MJ Shapter FM Henry RJ 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2006,112(2):358-365
Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access
to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised
equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised
a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show
here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal
SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence
tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic
probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed
to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95%
success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers
were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from
the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR
is highly successful for the development of robust SNP markers.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
9.
DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. 总被引:111,自引:19,他引:111
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Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR-based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative. 相似文献
10.
U Büyük Ö Ates L Dalyan B Müsellim G Öngen A Topal‐Sarıkaya 《Cell biochemistry and function》2010,28(4):274-277
Systemic sclerosis (SSc) is an autoimmune disease characterized by inflammation and fibrosis of the skin and visceral organs. Fibrosis associated with SSc is characterized by an increased synthesis of a wide range of extracellular matrix (ECM). TGF‐β is a pluripotent cytokine in a wide range of cell types. In particular it has been found to be a potent inducer of ECM protein synthesis and fibroblast migration. The TGF‐β1 gene is highly polymorphic and two signal sequence polymorphisms at codon 10 and codon 25 are linked to disease outcomes. In this study, we analysed two polymorphic sites of the TGF‐β1 gene, codon 10 and codon 25, in 43 Turkish SSc female patients with interstitial lung involvement and in 75 healty individuals by ARMS‐PCR. In our study no significant difference was found in codon 10, codon 25 genotype frequencies between patient with SSc and the control group (p = 0.676, 0.375, respectively). Our findings suggest that codon 10 and 25 polymorphism cannot be related with SSc for Turkish population. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
11.
目的:改进现有的检测表皮生长因子受体(EGFR)基因突变的荧光PCR法并开发出新的试剂盒,将其与直接测序法和ARMS法进行对比,验证该试剂盒用于临床诊断的敏感性、特异性和准确性。方法:收集2013年6月至2015年8月手术确诊的141例非小细胞肺癌(NSCLC)的石蜡包埋组织标本。采用盲法分别使用直接测序法、ARMS法和新试剂盒检测EGFR突变,比较新试剂盒与其他两种检测方法的差异,结果不一致时采用三种方法分别重复检验一次。结果:三种方法检测成功率均为100%,新试剂盒与直接测序法测得结果完全一致的比率达75.9%(107/141),在直接测序法测得的96例突变阳性中,92例在新试剂盒检测中得到验证(95.8%)。而直接测序法显示突变阴性的45例中,新试剂盒检测发现了23例突变阳性,两种检测方法的结果存在统计学差异(x2=40.745,P0.05)。与直接测序法进行比较,新试剂盒检测EGFR突变的敏感性、特异性分别为95.8%、48.9%,阳性预测值、阴性预测值分别为80.0%、84.6%,检测准确度为80.9%。以ARMS检测法为金标准,新试剂盒测得结果完全一致的比率达84.4%(119/141),两者的一致性比较好(K=0.749,P0.05),敏感性、特异性分别为94.1%、86.4%。结论:改进后EGFR基因突变检测的试剂盒在技术上较好地控制了检测结果的假阳性和假阴性,该检测方法较直接测序法具有更好的敏感性和准确性,与现有的ARMS法一致性较高。 相似文献
12.
We present an improvement of the inverse PCR method for the determination of end sequences of restriction fragments containing unknown DNA sequences flanked by known segments. In this approach, a short "bridge" DNA is inserted during the self-ligation step of the inverse PCR technique. This bridge DNA acts as primer annealing sites for amplification and subsequent direct sequencing. Successive PCR amplifications enable selective amplification of the unknown sequences from a complex mixture. Unlike previously described methods, our method does not require special materials, such as synthetic adapters or biotinylated primers that must be prepared each time to adapt the target. Furthermore, no complex steps such as dephosphorylation or purification are needed. Our method can save time and reduce the cost of cloning unknown sequences; it is ideal for routine, rapid gene walking. We applied this method to a GC-rich bacterial genome and succeeded in determining the end sequences of a 4.5-kb fragment. 相似文献
13.
Genome wide patterns of nucleotide diversity and recombination reveal considerable variation including hotspots. Some studies suggest that these patterns are primarily dictated by individual locus history related at a broader scale to the population demographic history. Because bottlenecks have occurred in the history of numerous species, we undertook a simulation approach to investigate their impact on the patterns of aggregation of polymorphic sites and linkage disequilibrium (LD). We developed a new index (Polymorphism Aggregation Index) to characterize this aggregation and showed that variation in the density of polymorphic sites results from an interplay between the bottleneck scenario and the recombination rate. Under particular conditions, aggregation is maximized and apparent mutation hotspots resulting in a 50-fold increase in polymorphic sites density can occur. In similar conditions, long distance LD can be detected. 相似文献
14.
Grønlund H Moen B Hoorfar J Radstrøm P Malorny B Rudi K 《Preparative biochemistry & biotechnology》2011,41(2):166-174
A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technology that both is highly accurate and enables multiplexing. A current bottleneck for direct genome analyses by minisequencing, however, is the sensitivity, since minisequencing relies on linear signal amplification. Here, we present SNPtrap, which is a novel approach that combines the specificity and possibility of multiplexing by minisequencing with the sensitivity obtained by logarithmic signal amplification by polymerase chain reaction (PCR). We show a SNPtrap proof of principle in a model system for two polymorphic SNP sites in the Salmonella tetrathionate reductase gene (ttrC). 相似文献
15.
Direct micro-haplotyping by multiple double PCR amplifications of specific alleles (MD-PASA) 总被引:5,自引:2,他引:3
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Analysis of haplotypes is an important tool in population genetics, familial heredity and gene mapping. Determination of haplotypes of multiple single nucleotide polymorphisms (SNPs) or other simple mutations is time consuming and expensive when analyzing large populations, and often requires the help of computational and statistical procedures. Based on double PCR amplification of specific alleles, described previously, we have developed a simple, rapid and low-cost method for direct haplotyping of multiple SNPs and simple mutations found within relatively short specific regions or genes (micro-haplotypes). Using this method, it is possible to directly determine the physical linkage of multiple heterozygous alleles, by conducting a series of double allele-specific PCR amplification sets with simple analysis by gel electrophoresis. Application of the method requires prior information as to the sequence of the segment to be haplotyped, including the polymorphic sites. We applied the method to haplotyping of nine sites in the chicken HSP108 gene. One of the haplotypes in the population apparently arose by recombination between two existing haplotypes, and we were able to locate the point of recombination within a segment of 19 bp. We anticipate rapidly growing needs for SNP haplotyping in human (medical and pharmacogenetics), animal and plant genetics; in this context, the multiple double PCR amplifications of specific alleles (MD-PASA) method offers a useful haplotyping tool. 相似文献
16.
Single copy nuclear polymorphic (scnp) DNA is potentially a powerful molecular marker for evolutionary studies of populations. However, a practical obstacle to its employment is the general problem of haplotype determination due to the common occurrence of heterozygosity in diploid organisms. We explore here a 'consensus vote' (CV) approach to this question, combining statistical haplotype reconstruction and experimental verification using as an example an indel-free scnp DNA marker from the flanking region of a microsatellite locus of the migratory locust. The raw data comprise 251-bp sequences from 526 locust individuals (1052 chromosomes), with 71 (28.3%) polymorphic nucleotide sites (including seven triallelic sites) and 141 distinct genotypes (with frequencies ranging from 0.2 to 25.5%). Six representative statistical haplotype reconstruction algorithms are employed in our CV approach, including one parsimony method, two expectation-maximization (EM) methods and three Bayesian methods. The phases of 116 ambiguous individuals inferred by this approach are verified by molecular cloning experiments. We demonstrate the effectiveness of the CV approach compared to inferences based on individual statistical algorithms. First, it has the unique power to partition the inferrals into a reliable group and an uncertain group, thereby allowing the identification of the inferrals with greater uncertainty (12.7% of the total sample in this case). This considerably reduces subsequent efforts of experimental verification. Second, this approach is capable of handling genotype data pooled from many geographical populations, thus tolerating heterogeneity of genetic diversity among populations. Third, the performance of the CV approach is not influenced by the number of heterozygous sites in the ambiguous genotypes. Therefore, the CV approach is potentially a reliable strategy for effective haplotype determination of nuclear DNA markers. Our results also show that rare variations and rare inferrals tend to be more vulnerable to inference error, and hence deserve extra surveillance. 相似文献
17.
The amplification refractory mutation system (ARMS) is routinely used for the identification of specific mutations within genomes. This PCR-based assay, although simple, is performed at a low-throughput scale, usually requiring gel-electrophoresis for the identification of specific mutations. We have applied the ARMS technology to a low-density microarray system to facilitate the needs of the medical clinic; high-throughput capabilities and ease-of-use. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene (DeltaF508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Amine-modified probes specific for both the wild-type and mutant forms of each mutation site were attached to glass substrates. Following hybridization of the PCR fragments to the attached probes (in a low-density microarray format), confirmation of the presence of specific sequences was achieved using a commercial scanner, as well as a fabricated low-cost fluorescent detector and applicable software. The novel combination of the ARMS and low-density microarray technologies allows for a high-throughput, simple means to rapidly identify multiple known mutations for many genetic diseases including cystic fibrosis. 相似文献
18.
Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers. 总被引:6,自引:0,他引:6
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We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3. 相似文献
19.
Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex. 相似文献
20.
Direct haplotyping of kilobase-size DNA using carbon nanotube probes 总被引:12,自引:0,他引:12
We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed. 相似文献