Start Codon Targeted (SCoT) Polymorphism: A Simple,Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.     

Molecular Characterization of High Plant Species Using PCR with Primers Designed from Consensus Branch Point Signal Sequences

A novel method is introduced for producing molecular markers in plants using single 15- to 18-mer PCR primers designed from the short conserved consensus branch point signal sequences and standard agarose gel electrophoresis. This method was tested on cultivated peanut and verified to give good fingerprinting results in other plant species (mango, banana, and longan). These single primers, designed from relatively conserved branch point signal sequences within gene introns, should be universal across other plant species. The method is rapid, simple, and efficient, and it requires no sequence information of the plant genome of interest. It could be used in conjunction with, or as a substitute for, conventional RAPD or ISSR techniques for applications including genetic diversity analysis, phylogenetic tree construction, and quantitative trait locus mapping. This technique provides a new way to develop molecular markers for assessing genetic diversity of germplasm in diverse species based on conserved branch point signal sequences.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.     

A simple method for the preparation of single-stranded polydeoxynucleotides of desired length.

A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.     

Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis

 Genes cloned from diverse plants for resistance to different pathogens have sequence similarities in domains presumably involved in pathogen recognition and signal transduction in triggering the defense response. Primers based on the conserved regions of resistance genes often amplify multiple fragments that may not be separable in an agarose gel. We used denaturing polyacrylamide-gel electrophoresis to detect PCR products of plant genomic DNA amplified with primers based on conserved regions of resistance genes. Depending upon the primer pairs used, 30–130 bands were detected in wheat, rice, and barley. As high as 47%, 40%, and 27% of the polymorphic bands were detected in rice, barley, and wheat, respectively, and as high as 12.5% of the polymorphic bands were detected by certain primers in progeny from a cross of the wheat cultivars ‘Stephens’ and ‘Michigan Amber’. Using F₆ recombinant inbred lines from the ‘Stephens’בMichigan Amber’ cross, we demonstrated that polymorphic bands amplified with primers based on leucine-rich repeats, nucleotide-binding sites and protein kinase genes, were inherited as single loci. Linkages between molecular markers and stripe rust resistance genes were detected. This technique provides a new way to develop molecular markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species. Received : 5 September 1997 / Accepted : 6 November 1997     

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.     

不同纯化方法处理DNA合成引物对PCR扩增效率的影响

对ＤＮＡ合成的幽门螺杆菌尿素酶引物ＨＰ１、ＨＰ２、ＨＰ３、ＨＰ４进行了几种不同的纯化试验,分别采用无水乙醇沉淀法、ＮＴ柱及聚丙烯酰胺凝胶电泳方法,对其相应的纯化收率,ＰＣＲ扩增效率作了比较及分析。琼脂糖凝胶电泳结果证实,以无水乙醇沉淀纯化方法的ＰＣＲ扩增效果较为理想。该方法操作简便、稳定高效、省时省力、成本低。为此建议用该法处理ＤＮＡ合成引物。     

Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR

A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

30个枣树种质资源遗传多样性的ISSR分析

取30个枣树品种进行ISSR分子标记分析,其扩增条带分别进行琼脂糖和聚丙烯酰胺凝胶电泳检测,以期获得不同产地品种之间的遗传多态性。从100条选择扩增的ISSR引物中筛选出17条扩增清晰、重复性和稳定性好的引物,选取其中8条扩增条带多态性强的引物进行遗传聚类分析。结果表明：（1）1％琼脂糖凝胶电泳和5％聚丙烯酰胺凝胶电泳检测扩增总条带数分别为72和127条,其中多态性条带分别为51条和113条,多态性条带比率（PPB）分别为70．8％和88．9％。（2）基于UPGMA软件对30个品种的遗传差异性分析表明,8个ISSR引物可以将枣树品种之间遗传差异明显区分开来。两种电泳检测方法相比较,聚丙烯酰胺凝胶电泳检测可获得较为精细的枣树品种间遗传图谱。其遗传相似系数范围在0．56～1．00之间,以0．62为最低遗传相似系数,可将30个枣树品种分成3个大类,6个亚类,为进一步研究枣树品种间分类、起源进化关系和分子辅助育种奠定基础。     

Papillomaviruses and herpesviruses as selected risk factors in the etiopathogenesis of cervix neck cancer. II. Use of PCR for isolating DNA of human papillomavirus and Herpes simplex

The aims of the study were to compare polymerase chain reaction PCR with nucleic acid hybridisation HC in the routine diagnosis of HPV infections. Smears collected for PCR were digested for 24 hours using proteinase K. After DNA extraction 174 samples were tested by PCR with human bglobin primers PG04-GH20. The PCR products were separated in 2% agarose gel electrophoresis stained with ethidium bromide. In 80.6% of the samples 256 base pair DNA fragments were observed in the gel in UV light. These samples were tested by PCR with HPV primers MY09-MY11. In 40% of the samples the presence of HPV DNA was confirmed. Next we carried out PCR using a mixture of two pairs of primers bglobin PG04-GH20 and HPV MY09-MY11. DNA for this study was extracted from 24 samples in which the presence of human DNA was not confirmed in the first PCR test and from 7 untested samples. In 21 cases HPV DNA was found to be present in gel electrophoresis. The presence of HPV DNA was confirmed in 44.75% of the samples.     

Application of fluorescence-based resistance gene analog analysis for genotyping plant genetic resources

Conventional approaches for detecting disease resistance gene analogs (RGAs) in plants are based on agarose gels or on polyacrylamide gel electrophoresis (PAGE) in combination with silver staining or radioactive labeling. A modified method for RGA analysis has been developed by using fluorescence-labeled primers for PCR amplifications. The amplified fragments are detected by denaturing PAGE using an automated laser fluorescence DNA sequencer and analyzed by fragment analysis software. This technique is not limited to specific plant species and is suitable for high-throughput genotyping plant genetic resources. We demonstrate here the efficiency of this method for comparison of RGA patterns in diverse plant species and for genotyping of natural populations of the wheat progenitor, Triticum dicoccoides. Revisions requested 8 October 2004; Revisions received 15 November 2004     

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

Summary DNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.     

Sexing the Sciuridae: a simple and accurate set of molecular methods to determine sex in tree squirrels, ground squirrels and marmots

We determined the sequence of the male-specific minor histocompatibility complex antigen (Smcy) from the Y chromosome of seven squirrel species (Sciuridae, Rodentia). Based on conserved regions inside the Smcy intron sequence, we designed PCR primers for sex determination in these species that can be co-amplified with nuclear loci as controls. PCR co-amplification yields two products for males and one for females that are easily visualized as bands by agarose gel electrophoresis. Our method provides simple and reliable sex determination across a wide range of squirrel species.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．     

Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.