A novel set of microsatellite marker loci linkage-mapped in the house musk shrew, Suncus murinus.

Ten microsatellite DNA loci developed for the white-toothed shrew (Crocidura russula) were tested for PCR amplification and for utility in linkage studies in the house musk shrew, Suncus murinus. Four primer pairs successfully yielded PCR amplicons and showed polymorphism between two mutant strains, BAN-kc,oeb and WZ. Cloning and sequencing of the PCR amplicons of all the four loci confirmed the presence of microsatellite sequences. Alleles segregating in an F2 resource population constructed from the two strains ranged between two and five. Linkage analysis of the four loci together with 18 other polymorphic markers and three mutant loci resulted in five linkage groups containing three newly mapped microsatellite loci. This study reports the first microsatellite markers being registered in this species.     

Isolation and characterization of 27 polymorphic microsatellite loci for the common snook,Centropomus undecimalis

Here we describe 32 di‐, tri‐ and tetranucleotide microsatellite loci isolated by PIMA, a polymerase chain reaction (PCR)‐based procedure, for the common snook (Centropomus undecimalis). Five loci were monomorphic, and the remaining loci averaged 6.7 alleles per locus in a sample of 45 common snook. For polymorphic loci, expected heterozygosities ranged from 0.02 to 0.91 (mean = 0.538). Significant departures from Hardy–Weinberg equilibrium expectations occurred in two loci. Exact tests for genotype disequilibrium gave evidence for linkage at one pair of loci. Many cross‐species primer assays yielded PCR fragments of the expected size for 11 species of Centropomus and two species of the confamilial genus Lates.     

Rapid classification of partial waxy wheats using PCR-based markers.

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

筛选适用于小型猪遗传检测的微卫星位点

目的筛选用于封闭群小型猪遗传检测的微卫星位点。方法从资料和GenBank中选取扩增效果好、等位基因多、均匀分布于小型猪18条常染色体和X染色体上的100个微卫星位点,合成引物,对封闭群小型猪基因组进行PCR扩增及条件优化。PCR产物采用琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和STR扫描技术进行分析和比较,选择多态性好的位点。结果筛选出32个分布于不同染色体且等位基因多的微卫星位点。结论筛选出了应用于封闭群小型猪遗传检测的微卫星位点。     

Generating single-copy nuclear gene data for a recent adaptive radiation

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.     

种间转移扩增法筛选长爪沙鼠微卫星位点

目的筛选长爪沙鼠新的微卫星位点,为长爪沙鼠遗传分析提供遗传标记物。方法从GenBank中随机选取小鼠微卫星位点引物536对,用这些引物对长爪沙鼠基因组DNA扩增,将阳性目的条带进行序列分析,找出符合微卫星序列特征的短串联重复序列。结果 536对小鼠微卫星引物在长爪沙鼠基因组中扩增出了313个阳性条带,经序列分析,确定130个长爪沙鼠微卫星位点;其中完美型位点占80.77%(105/130),不完美型位点占19.23%(25/130),与小鼠同源性为24.3%(130/536)。将筛选出的微卫星位点在GenBank中注册,注册号从GU562694到GU562823。结论小鼠和沙鼠的微卫星位点具有较高的同源性,用小鼠的微卫星位点引物直接扩增长爪沙鼠基因组DNA可有效地筛选出长爪沙鼠微卫星位点。     

Highly accurate analysis of heterozygous loci bysingle cell PCR.

Single cell PCR is a powerful method for determining the genetic properties of individual cells. In the instance of heterozygous loci, however, preferential amplification of one allele can lead to allele drop out (ADO) of the other allele. Fortunately ADO does not occur in all amplifications, and is usually random when it does occur, with both alleles being equally susceptible to drop out. Therefore pooling of results from multiple independently amplified cells should greatly improve the analysis of diallelic loci, and the misdiagnosis rate of diallelic loci should decrease exponentially with the number of cells analysed. We have shown that this is true and that multiplex PCR allows for the simultaneous identification of a cell in a mixture of cells using microsatellite loci known to be informative, and accurate genotyping at other loci. This approach has practical applications to non-invasive prenatal diagnosis where small numbers of fetal cells in the presence of maternal cells must be both identified and genotyped at loci involved in genetic disease.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci

Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions.     

Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm: experimental design considerations

The polymerase chain reaction (PCR) makes it possible to rapidly generate a very large number of copies of a specific region of DNA. Application of PCR to individual human sperm cells permits the typing of a large number of independent male meiotic events. If the donor male is heterozygous at three loci, sperm typing using PCR will permit ordering of loci in a manner analogous to classical methods of experimental genetics. Sequential analysis of trios of loci by sperm typing will provide a very accurate means of ordering any number of tightly linked loci. Here, we describe experimental design and sample-size issues raised by the application of sperm typing by PCR for mapping human chromosomes, and we demonstrate that sperm typing will be an efficient method for generating fine-structure human genetic maps.     

利用电子PCR分析草菇基因组SSR标记多态性

【目的】利用电子PCR分析草菇基因组SSR标记多态性,并通过PCR验证分析结果的可靠性。【方法】利用MISA程序定位草菇基因组SSR位点并结合Primer3.0程序设计SSR分子标记引物,运用电子PCR进行SSR分子标记多态性分析,基于分析结果进行PCR验证。【结果】随机选取658对SSR引物在草菇同核体菌株V23-1和PD19中进行真实PCR检测,结果表明28.6%的SSR引物具有多态性。数据分析表明,如果SSR标记来源于电子PCR产物长度没有差异的类型,仅4.8%的SSR引物在真实PCR中表现出多态性;如果SSR标记来源于电子PCR产物长度差异大于或者等于3 bp的类型,其中至少48.3%的SSR引物在真实PCR中表现出多态性。【结论】PCR验证结果表明利用电子PCR可以提高SSR多态性引物的筛选效率。     

恒河猴群微卫星DNA多态性的分析

目的　确立一种对恒河猴群个体的遗传物质进行准确可靠、快速简便的遗传检测方法。方法　利用聚合酶链反应 (PCR)扩增技术对 2 0只恒河猴群个体间进行了DNA多态性的分析。结果　筛选出 9个微卫星DNA位点具有显著多态性 ,4个微卫星DNA位点没有多态性 ,还有 2个位点等位基因数目较少。结论　利用这些多态性微卫星位点建立一种对恒河猴群个体进行有效、准备可靠、快捷简便的遗传背景监测方法。     

Microsatellite loci from the house wren (Troglodytes aedon)

We cloned seven microsatellite loci from house wrens (Troglodytes aedon) using a biotin enrichment protocol. Starting with fragments generated using DOP–PCR, fragments containing microsatellite motifs AC and AAC were captured using biotinylated probes and streptavidin coated magnetic particles. Captured fragments were cloned into plasmids; prior to sequencing, the plasmids were screened for microsatellites using a simple PCR approach. Five of the loci showed variation in a sample of nine individuals.     

Development of a touch-down multiplex PCR method for simultaneously rapidly detecting three novel insertion/deletions (indels) within one gene: an example for goat GHR gene

Abstract

The multiplex polymerase chain reaction (PCR) method is fast and accuracy for screening several polymorphism loci in a single reaction in large samples. Therefore, this study aimed to identify three novel insertion/deletion (indel) loci in goat growth hormone receptor (GHR) gene by using multiplex PCR method and the genotypes in three indel loci are identified in 918 individuals from three Chinese goat breeds, as well as to evaluate its associations with growth traits. After validating the accuracy by taking the conventional PCR method, the concordance between these two methods was 100%. Moreover, P14 locus polymorphism was significantly associated with six growth traits of Hainan black goats. Briefly, an economic multiplex PCR method is presented to simultaneously and accurately detect three indel polymorphisms within goat GHR without the use of any special equipment, thus improving the experimental efficiency and fast obtaining the experimental result for indel genotyping.     

Characterization of seven polymorphic microsatellite loci in the Common Loon (Gavia immer)

We describe polymerase chain reaction (PCR) primers and conditions to amplify seven microsatellite DNA loci isolated from the Common Loon (Gavia immer). The PCR primers were tested on 83 individuals from 10 locations in North America, including breeding, migration stopover, and wintering areas. Between two and seven alleles were observed to segregate at the seven microsatellite loci, with observed heterozygosities ranging from 0.048 to 0.695.     

Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major

Thirteen sets of polymerase chain reaction (PCR) primers were designed to amplify microsatellite loci identified in the genome sequence of Leishmania major. Polymorphisms were detected in L. major at all loci. In Leishmania donovani only two of these loci were informative for classification purposes with this data set. The PCR products of all loci from one L. donovani strain were sequenced and it was found that the number of repeats in the microsatellite loci were either substantially reduced with respect to L. major or absent altogether. Consequently it is unlikely to be possible to use the genome sequence of L. major to identify polymorphic microsatellite loci in other Leishmania species.     

Technical advance: single pollen typing combined with laser-mediated manipulation

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.     

Characterization of novel microsatellites and development of multiplex PCR for large‐scale population studies in wild cherry,Prunus avium

Primers were developed for 14 microsatellite or simple sequence repeat (SSR) loci identified from a Prunus avium‘Charger’ genomic DNA library. In a survey of 16 wild cherry accessions 10 of the loci revealed polymorphisms of between two and six alleles. The remaining loci were found to be monomorphic. Seven polymorphic loci identified in this study and four polymorphic loci previously reported in sweet cherry were mapped and found to be unlinked. Two multiplex polymerase chain reactions (PCR) were optimized to enable the characterization of all 11 unlinked, polymorphic SSR loci.