Short tandem repeat analysis in Japanese population

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.     

Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.     

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.     

A rapid polymerase chain reaction method for identifying fixed specimens.

DNA profiling analysis of preserved tissue samples was achieved by using an extraction method which incorporated the use of a chelating resin "Chelex" to chelate inhibitory polyvalent metal ions. An automated sequencer was used to estimate sizes of low molecular weight trimeric and tetrameric microsatellites labelled with fluorescent dyes. The entire analysis can be completed within 48h. Probabilities of chance association using two microsatellites were calculated as ranging between 10(-2) and 10(-4). The methods described have considerable potential for use in routine genetic analysis of preserved samples.     

Genetic polymorphism of 13 non‐CODIS STR loci in three national populations from China

The aim of this study was to investigate a 13 non‐CODIS STR loci database using three national populations from China. A new multiplex PCR system that simultaneously amplified 13 loci in the same PCR reaction was developed. This multiplex system included the 13 STR markers (D3S2402, D3S2452, D3S1766, D3S4554, D3S2388, D3S3051, D3S3053, D4S2364, D4S2404, AC001348A, AC001348B, D17S975, and D17S1294), which were successfully analyzed by using 441 DNA samples from three national populations in China (154 Mongol, 177 Kazakh, and 110 Uigur). Allele frequencies and mutation rates of the 13 non‐CODIS STR loci were investigated. A total of 4–10 alleles at each locus were observed and altogether 84, 88, and 87 alleles for the all selected loci were found in the Mongol, Kazakh, and Uigur, respectively. Eight mutations were detected from the 13 selected loci in 9880 meioses in kinship cases. These results indicate that this multiplex system may provide significant polymorphic information for kinship testing and relationship investigations.     

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer

A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.     

Development of multiplex PCR system with 15 X-STR loci and genetic analysis in three nationality populations from China

The aim of this study is to develop a new multiplex PCR system that simultaneously amplifies the 15 X-chromosome short tandem repeats (X-STRs) loci in the same PCR reaction, and to obtain the 15 X-STR loci database in three nationality populations from China. This multiplex system includes DXS7133, DXS6801, DXS981, DXS6809, DXS7424, DXS6789, DXS9898, DXS7132, GATA165B12, DXS101, DXS10075, DXS6800, GATA31E08, DXS10074, and DXS10079, which were successfully analyzed on 1251 DNA samples (670 males and 581 females) from Guangdong Han population, Xinjiang Uigur and Kazakh. The allele frequencies and mutation rates of the 15 loci were investigated, and the allele frequency distribution among different populations was compared. A total of 6-17 alleles for each locus were observed and altogether 170 alleles for all the selected loci were found. Thirteen cases with mutation of the above loci were detected in 11,850 meioses. Pairwise comparisons of the allele frequencies distribution showed significant differences in most loci among different populations. The results indicate that this multiplex system may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Single laboratory validation of a microsatellite marker-based method in tomato variety identification

The objective of this study was to develop a systematic and flexible method for assembling multiplex simple sequence repeat marker panels for high-throughput genome analysis in the tomato, Solanum lycopersicum, for varietal identification and to demonstrate the technical viability of these genetic markers for use in the enforcement of U.S. Department of Agriculture marketing order-based identity preservation programs. GeneMapper, a semiautomated software tool, was used for designing multiplex panels, allele identification, and polymorphism pattern evaluation of diverse tomato cultivars. Semiautomated genotyping was performed on a set of 12 microsatellite markers providing genome-wide coverage of the tomato chromosomes. Microsatellites were detected with fluorescently labeled primers grouped into five multiplex panels, and each primer pair was assessed in replicated trials for reliability of allele size estimates. Allele sizes for each locus were compared, and a database for 34 tomato varieties was developed. The microsatellite marker set identified distinct allelic peaks and unique genetic fingerprints for each of the studied tomato varieties. A "blind testing" exercise with UglyRipe and Vintage Ripe tomato varieties, using the above set of markers and database, further established the usefulness of these microsatellite markers for tomato commodity marketing order enforcement.     

Multiple genotyping based on multiplex PCR and microarray

The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors.Herein,we have described a multiple genotyping method based on magnetic enrichmentmultiplex PCR (MEM-PCR) and microarray technology.Monodisperse magnetic beads were fabricated and modified with streptavidin.Four loci on two genes (M235T and A-6G loci on AGT gene,A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP).Target sequences of these SNP loci were amplified using Cy3-labeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB).Four pairs of NH₂-labeled probes,corresponding to each locus,were fixed on CHO-modified glass slide by covalent binding.Hybridization between target sequences and probes was performed under suitable conditions.The spotting locations on microarray and the ratio of fluorescence intensity,produced by different loci,were used to distinguish the SNP genotypes.Finally,three of gastric cancer samples were collected and genotyping analysis for these four SNP loci was carried out successfully simultaneously by this method.     

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.     

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.     

Simultaneous analysis of multiple PCR amplicons enhances capillary SSCP discrimination of MHC alleles

Major histocompatibility complex (MHC) genotyping still remains one of the most challenging issues for evolutionary ecologists. To date, none of the proposed methods have proven to be perfect, and all provide both important pros and cons. Although denaturing capillary electrophoresis has become a popular alternative, allele identification commonly relies upon conformational polymorphisms of two single‐stranded DNA molecules at the most. Using the MHC class II (β chain, exon 2) of the black kite (Aves: Accipitridae) as our model system, we show that the simultaneous analysis of overlapping PCR amplicons from the same target region substantially enhances allele discrimination. To cover this aim, we designed a multiplex PCR capable to generate four differentially sized and labeled amplicons from the same allele. Informative peaks to assist allele calling then fourfold those generated by the analysis of single PCR amplicons. Our approach proved successful to differentiate all the alleles (N=13) isolated from eight unrelated birds at a single optimal run temperature and electrophoretic conditions. In particular, we emphasize that this approach may constitute a straightforward and cost‐effective alternative for the genotyping of single or duplicated MHC genes displaying low to moderate sets of divergent alleles.     

Rapid individual identification by minisatellite variant repeat (MVR)-PCR at D1S8 locus using "exponential law".

We describe an efficient and simple minisatellite variant repeat mapping by PCR (MVR-PCR) method based on the assignment of the tandem array of 29 bp repeating units into a-type, t-type and 0-type (a rarely appearing unamplified unit), from the first repeat unit position (code position 0) of 293 bp in D1S8 locus. After microchip electrophoresis of PCR product amplified from the target DNA of a human hair root, each rung of the ladder at the position of 293 + 29 n bp (n: code position) was detected and the type of repeating unit was determined, i.e., aa, a0, tt, t0, at and 00. The peak area of the rungs from PCR product decreased with increase in code position. We found for the first time that the logarithmic plots of the peak area against the code position showed a linear relationship, which implied that peak areas decrease exponentially. The present method was successfully applied to identify 37 individuals using only a hair root as a biological specimen. This "exponential law" is expected to be an effective tool in forensic science.     

A simple and affordable method for high-throughput DNA extraction from animal tissues for polymerase chain reaction

We have developed a very simple and inexpensive method for high-throughput DNA extraction from animal tissues. The procedure contains three steps (digestion, heating, and centrifugation) and it is compatible with the 96-well plate format commonly used in polymerase chain reaction (PCR) amplifications. The duration for processing a plate is about 1.5 h; therefore, one researcher can isolate DNA from up to 1000 samples during a single workday. A small piece of tissue (ca. 10-20 mg) yields enough template for at least 50-70 PCR amplifications, as demonstrated by using the processed samples as templates successfully for long distance PCR, multiplex PCR, and randomly amplified polymorphic DNA (RAPD) assay. The application of our method is expected to facilitate studies that require high-throughput DNA isolation for PCR amplification, such as genotyping by microsatellites for mapping and genetic diversity studies, as well as mutant screening in zebrafish.     

Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed-side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel-based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.     

Development of a body fluid identification multiplex via DNA methylation analysis

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers – BCAS4, CG06379435, VE_8, and ZC3H12D – were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.     

Computer analysis of automated Edman degradation and amino acid analysis data

Computer programs are described that allow facile analysis of data from a protein sequencer and amino acid analyzer. The sequencer program provides automated sequence interpretation while requiring minimal user interaction. The program serves as a powerful aid in deciphering mixture sequences and allows routine monitoring of sequencer performance. The computer program for amino acid analysis data provides the following calculations: mole percent, protein concentration and residues per mole with comparison between theoretical and calculated values. A plot of molecular weight versus deviation from integer values is calculated providing a measure of peptide or protein purity.     

A new autosomal STR nineplex for canine identification and parentage testing

A single multiplex PCR assay capable of simultaneously amplifying nine canine‐specific autosomal STR markers (FH3210, FH3241, FH2004, FH2658, FH4012, REN214L11, FH2010, FH2361 and the newly described C38) was developed for individual identification and parentage testing in domestic dogs. In order to increase genotyping efficiency, amplicon sizes were optimized for a 90–350 bp range, with fluorescently labelled primers for use in Applied Biosystems, Inc., platforms. The performance of this new multiplex system was tested in 113 individuals from a case‐study population and 12 random dogs from mixed‐breed origin. Co‐dominant inheritance of STR alleles was investigated in 101 father, mother and son trios. Expected heterozygosity values vary between 0.5648 for REN214L11 and 0.9050 for C38. The high level of genetic diversity observed for most markers provides this multiplex with a very high discriminating power (matching probability=1.63/10¹⁰ and matching probability among siblings=4.9/10³). Allele sequences and a proposal for standardized nomenclature are also herein presented, aiming at implementing the use of this system in forensic DNA typing and population genetic studies. This approach resulted in an optimized and well‐characterized canine DNA genotyping system that is highly performing and straightforward to integrate and employ routinely. Although this STR multiplex was developed for use and tested in a case‐study population, the Portuguese breed Cão de Gado Transmontano, it proved to be useful for general identification purposes or parentage testing.