A strategy for single site PCR amplification of dsDNA: priming digested cloned or genomic DNA from an anchor-modified restriction site and a short internal sequence

Amplification of dsDNA by polymerase chain reaction (PCR) has been limited to those instances in which segments of known sequence flank the fragment to be amplified. A strategy for the PCR amplification of cloned or genomic dsDNA that necessitates sequence information from only a single short segment (single site PCR) has been devised. The region of known sequence may be located at any position within or adjacent to the segment to be amplified. The basic procedure for amplification consists of 1) digestion of dsDNA with one or more restriction enzymes, 2) ligation with a universal anchor adaptor and 3) PCR amplification using an anchor primer and the primer for the single site of known sequence. The anchor adaptor is designed in such a way as to facilitate the amplification of only those fragments containing the sequence of interest. We have demonstrated the utility of this technique by specifically amplifying and directly sequencing antibody variable region genes from cloned dsDNA and from genomic DNA.     

Rapid detection of maize DNA sequence variation.

The allele-specific polymerase chain reaction (ASPCR) has been used to determine the genotype of maize lines at two loci, wx and NPI288. The ASPCR method uses allele-specific oligonucleotide primers in PCR amplifications to amplify and discriminate simultaneously between polymorphic alleles. The success of this technique relies on the specific failure of PCR to amplify with primers that do not perfectly match the DNA sequence of one of the allelic variants. Amplification results were evaluated by dot-blot hybridization using an alkaline-phosphatase-coupled probe. The technique's speed, accuracy, sensitivity, and high throughput make it valuable for plant-breeding applications.     

Development of sequence tagged microsatellites (STMs) for the barley scald pathogen Rhynchosporium secalis

A rapid and cost efficient technique was developed and used to generate 168 sequence tagged microsatellites (STMs) in the barley scald pathogen Rhynchosporium secalis. Sixty‐two STMs, amplifying 66 loci, revealed a high level of polymorphism among a diverse set of 16 Australian isolates. Each locus revealed two to nine alleles (average 4 ± 1.82), and a gene diversity measure of 0.54 was obtained. This technique not only halved the cost of marker development compared to traditional methods, but substantially reduced the cost of performing fluorescence‐based microsatellite assays. These STMs provide a powerful tool for genetic studies in R. secalis.     

Shotgun haplotyping: a novel method for surveying allelic sequence variation

Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.     

A molecular approach to the identification of S-alleles in Brassica oleracea

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-allele present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.     

The identification of sex in the starling Sturnus vulgaris using a molecular DNA technique

Female birds can be identified through the presence of a W-chromosome. We describe a procedure for amplifying a W-linked DNA marker in the starling (Sturnus vulgaris) by the polymerase chain reaction (PCR) so allowing the diagnosis of sex in this species. The technique is sensitive, allowing even the smallest chicks to be sexed from a blood sample. The method possesses a positive internal control to ensure accuracy. It is also applicable to the spotless starling (S. unicolor) but not to two bird species outside the genus. The nucleotide sequence of the female-specific PCR product is given.     

AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.     

The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on pinus root systems

Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species.     

扩增未知序列DNA片段的PCR技术研究进展

1985年,Mullis等人发明了PCR技术,短短十余年间,这一技术得到迅速发展和应用,已由扩增已知基因发展到扩增未知基因。本文旨在介绍利用PCR技术扩增未知序列DNA片段的最新进展及这些技术在基因克隆研究中的应用。     

Length and sequence variation in the apolipoprotein B intron 20 Alu repeat.

We have developed a single-stranded conformation polymorphism (SSCP) protocol for typing both sequence and length variations in an Alu element located in intron 20 of the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction (PCR), we simultaneously amplified and isotopically labeled the apo B intron 20 Alu. The Alu tail, which is composed of two arrays of variable numbers of tandem repeats, (TTTX)y (X = A or G) and (T)z, was separated from the rest of the PCR product by restriction enzyme digestion with PstI. Length variation in the Alu tail (IN20-REP) was thus separated from sequence variation in the Alu body (IN20-SEQ), rendering the SSCP patterns both eaiser to interpret and more informative. In a sample of 242 unrelated individuals from Nancy, France, we observed 11 SSCP alleles at the IN20-SEQ locus that differed only in sequence. At the IN20-REP locus, we observed 7 alleles that differed in both sequence and length. All alleles at both loci were subcloned and sequenced. One additional allele that did not undergo a detectable mobility shift in SSCP gels was uncovered at each locus during sequencing of the SSCP alleles. The additional IN20-SEQ allele was typed by restriction enzyme digestion. Although the number of IN20-SEQ and IN20-REP alleles was large, most were uncommon; the three most common alleles at each locus represented more than 94% of those sampled. We also typed the children of the 242 unrelated French individuals, enabling verification of the Mendelian segregation of the two loci and construction of haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid determination of multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis

This report presents a new method for identifying multi-locus sequence types of Listeria monocytogenes by microtemperature-gradient gel electrophoresis (μ-TGGE). Genomic comparison of L. monocytogenes serovar 1/2a strains EGD-e and F6854 allowed selection of novel polymorphic sequences lmo0386 and lmo0428 as optimum regions for μ-TGGE analysis, in addition to the previously identified lmo0297 gene. Sequence analysis of a total of 48 standard strains revealed that the strains could be grouped into 7 (lmo0386), 8 (lmo0428) and 12 (lmo0297) sequence types. The PCR products from 2, 4 and 4 sequence types of the lmo0386, lmo0428 and lmo0297 genes were selected as marker alleles, and μ-TGGE analysis of the mixture revealed adequate band separation on a single gel. Furthermore, the primer sets could be successfully mixed in a single tube for multiplex PCR, yielding a rapid and easy strategy for sequence type identification. For practical application, multiplex PCR was performed with Cy3-labeled primers against a sequence type-unknown sample isolated from meat. The resulting products were mixed with Cy5-labeled products of marker alleles whose sequence types were known, and μ-TGGE analysis was performed. Overlapping Cy3 and Cy5 patterns allowed identification of the sequence types at all 3 loci on a single gel. Moreover, the μ-TGGE analysis step took only 9 min. Thus, this novel method of multiplex PCR followed by μ-TGGE analysis could prove useful as a rapid and discriminative tool for tracing the strain types, contamination routes and sources of L. monocytogenes.     

Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay.

Single nucleotide substitutions and unique insertions/deletions are the most common form of DNA sequence variation and disease-causing mutation in the human genome. Because of the biological and medical importance of these variations, a wide array of methods have been developed for their typing. We have applied an approach that combines the amplification of polymorphic regions by the polymerase chain reaction (PCR) with a system for typing diallelic variants using an oligonucleotide ligation assay (OLA). In this report, we describe a significant advance in this technology that permits the typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors. We demonstrate the specificity, sensitivity and ease of data interpretation with this system. Furthermore, we show that multiplex PCR/OLA not only increases the throughput of DNA typing but also increases its accuracy in typing diallelic sequence variations using an approach that can be broadly applied for human genome analysis (in evaluating genotype/phenotype links), in typing infectious agents and in forensic analysis.     

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.     

Analysis of the BglI restriction fragment length polymorphism in the human factor VIII gene using “virtual PCR”– a novel approach employing the polymerase chain reaction in the absence of sequence information for the locus

The BglI restriction fragment length polymorphism (RFLP) of the human factor VIII (FVIII) gene is potentially useful in linkage studies in haemophilia A. The sequence at the RFLP locus is not known, therefore it is not amenable to analysis by the polymerase chain reaction (PCR) and Southern blotting is required. We present a novel approach for analysis of the BglI RFLP using the PCR targeted to known sequence downstream in exon 26 of the FVIII gene. Briefly, the size of the genomic restriction fragment carrying the PCR target depends upon whether the RFLP site is present or absent. If fragments of the required size are isolated from a genomic digest and used as substrates in the exon 26 PCR, the generation of a product in one or other fraction indicates the upstream RFLP status. We have called this approach “virtual PCR”, since PCR is used to obtain information about the RFLP without amplifying the locus itself. Received: 23 January 1996 / Revised: 18 March 1996     

Rapid estimation of spoilage bacterial load in aerobically stored meat by a quantitative polymerase chain reaction

We report a quantitative PCR which utilizes primers from a conserved 23S rDNA sequence identified in nine different spoilage bacteria commonly present in meat. The PCR detected the spoilage bacteria by amplifying a specific 207 bp sequence from their chromosomal DNA. Quantification of PCR product by electrochemiluminescence revealed that the concentration of the amplified product was dependent on cycle number and the initial number of bacteria present in the sample. Statistical analysis of the results indicated a correlation coefficient of 0.94 (P < 0.001) between aerobic plate count and QPCR luminosity units.     

PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower

Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.     

Rapid amplification of 5' complementary DNA ends (5' RACE)

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.