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PubMed1996

Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification.

Short tandem repeat (STR) loci are routinely employed for individual identification. WE have examined the performance and reproducibility of a highly informative co-amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50-500 pg, partial profiles were produced, but no allelic drop-out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop-out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.

Buffers↗
PubMed1996

High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers.

Short tandem repeat regions (STRs) from the polymorphic loci VWFA, THO1, TPO and CSF were amplified by the multiplex polymerase chain reaction (PCR) and analyzed by capillary array electrophoresis with fluorescence detection of energy transfer (ET) labels. The fluorescent ET primers are labeled with one fluorescein at the 5' end and a second fluorescein at the position of the 7th or 9th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). M13 A-track sequencing fragments, used as an internal sizing standard, were generated with a universal primer that has a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 11th (modified) base to produce fragments fluorescing in the red (> 590 nm). The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations were performed on arrays of hollow fused silica capillaries filled with denaturing and replaceable hydroxyethyl cellulose sieving matrices. Separations were complete in less than 50 min, and single base resolution as well as reproducible STR sizing was achieved. The relative standard deviation in sizing was below 0.6%. This work establishes the feasibility of high-resolution, high-speed and high-throughput STR typing of single-stranded DNA fragments using capillary array electrophoresis.

Bacteriophage M13↗
PubMed1996

DNA typing of a polymerase chain reaction amplified D1S80/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix.

In this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples, samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.

Alleles↗
PubMed1998

Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection.

A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.

DNA, Single-Stranded↗
PubMed1998

Multiplexed short tandem repeat polymorphisms of the Weber 8A set of markers using tailed primers and infrared fluorescence detection.

Short tandem repeat polymorphism (STRP) markers have become important reagents for mapping genetic diseases. These markers are available as screening sets, which are located in all chromosomes at discrete intervals, allowing the entire genome to be analyzed. Mapping studies that include many individuals in the analysis necessitate the production of large numbers of genotypes. In an effort to increase the efficiency and lower the cost of using these STRP screening sets, we have divided the amplification primers of the Weber 8A screening set into groups that can be amplified in single polymerase chain reaction (PCR) amplification reactions, resulting in a reduction of both time and cost. Fluorescently-labeled amplification products were produced using a three primer reaction. The forward STRP amplification primer for each marker contained a 19 bp sequence at the 5' end. A fluorescently-labeled primer, with a sequence identical to the 19 bp tail, was added to the amplification reaction as the sole source of fluorescent label. The STRP banding pattern is detected using an automated fluorescent DNA sequencer. Use of this multiplexed genomic screening set should greatly enhance the mapping of human disease loci.

DNA Primers↗
PubMed2003

Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually related tissues in hierarchical clustering algorithm analysis.

We have developed an experimental system to study the expression of 68 human glycosyltransferase genes. Using this system, we examined the expression of those genes in 27 different tissues by the technique which we named systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR). The panoramic view of a total of 1836 (68 x 27) expression data demonstrates that some glycosyltransferase genes are differentially expressed whereas some others are ubiquitously expressed. The data gathered provide more information on glycosyltransferase gene expression in tissues than any other paper published, and surpass in quantity all the information combined from previous publications. Although the expression profiling of glycosyltransferase genes alone may not directly explain the repertoires of oligosaccharides synthesized, it is an important step toward a better understanding of the gene expression network involved in oligosaccharide synthesis/degradation. Our modestly high-throughput gene expression study and the data analysis using a hierarchical clustering algorithm have allowed us to investigate the correlation between tissues and glycosyltransferase gene expression. Similar patterns of glycosyltransferase gene expression were observed in functionally and anatomically related tissues. All, but one, sexually related tissues formed a cluster in a tissue dendrogram, suggesting the involvement of sex hormones in the transcriptional control of many glycosyltransferase genes. Once established, the SM RT-PCR is cost- and time-efficient and requires small amounts of RNA as template. It is especially useful for the simultaneous analyses of multiple samples. Because of its simple design, the SM RT-PCR may offer an easy alternative in studying the expression of many other families of genes, as well as groups of related/unrelated genes, in various biological phenomena.

Algorithms↗
PubMed2004

Gene expression analysis of an integrin family of genes by systematic multiplex reverse transcription-polymerase chain reaction.

We have established the systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR) system of 26 members of the integrin family of genes, and used this system to investigate their expression in 25 different kinds of human adult tissues. A hierarchical clustering analysis of the tissue expression data illustrated clustering of functionally related tissues. Although the correlation was weak, bone marrow, thymus, and spleen were clustered, potentially confirming the role of certain integrin molecules in the immune response. We also investigated changes in integrin gene expression in cancer. Several differences were observed between normal and tumor, as well as between normal and cancer cell lines. The SM RT-PCR experiments for the study of alternative splicing showed that most of the integrin genes analyzed exhibited fixed ratios of differentially spliced transcripts probably due to a tissue-independent mechanism of splicing predetermined by the nucleotide sequences around the splicing donor and acceptor sites.

Animals↗
PubMed2004

Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications.

Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution-phase isoelectric focusing is an effective alternative to sucrose-gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence-based multiplexing dye technologies greatly extend the capacity of SDS-polyacrylamide gel electrophoresis with respect to the investigation of proteome-wide changes in protein expression and post-translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two-dimensional gel electrophoresis.

Animals↗
PubMed2005

Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets.

Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA.

Cell Line, Tumor↗
PubMed2005

Automatic analysis of multiplex ligation-dependent probe amplification products (exemplified by a commercial kit for prenatal aneuploidy detection).

For use in routine prenatal diagnostics, we developed software and methods for automatic aneuploidy detection based on a commercial multiplex ligation-dependent probe amplification (MLPA) kit. Software and methods ensure a reliable, objective, and fast workflow, and may be applied to other types of MLPA kits. Following CE of MLPA amplification products, the software automatically identified the peak area for each probe, normalized it in relation to the neighboring peak areas of the test sample, computed the ratio relative to a reference created from normal samples, and compensated the ratio for a side effect of the normalization procedure that scaled all chromosomally normal DNA peak areas slightly up or down depending on the kind of aneuploidy present. For the chromosomes 13, 18, 21, X, and Y, probe reliability weighted mean ratio values and corresponding SDs were calculated, and the significance for being outside a reference interval around ratio 1.0 was tested. p < or = 1% suggested aneuploidy and 1 < p < or = 5% suggested potential aneuploidy. Individual peaks, where the normalized area was situated more than 4 SD from the corresponding reference, suggested possible partial deletion or gain. Sample quality was automatically assessed. Control probes were not required. Having used the software and methods for two years, we conclude that a reliable, objective, and fast workflow is obtained.

Aneuploidy↗
PubMed2006

DNA sequencing and multiplex STR analysis on plastic microfluidic devices.

The ability of plastic microfluidic devices with separation channel lengths of 6, 10 or 18 cm to perform high-quality and high-performance ssDNA analysis was evaluated. Specifically, four-color DNA sequencing separation of a terminator sequencing standard using replaceable, urea-denaturing linear polyacrylamide (LPA) solution as a sieving matrix, yielded read lengths of 410 bases in 15 min with base calling accuracy of 99.2% on a 6-cm device, and 640 bases in 35 min with accuracy of 98.0% on a 18-cm device. A two-color sizing analysis of four-locus (CSF1PO, TPOX, TH01, vWA) short tandem repeats (STRs) allelic ladder on a 10-cm device indicated a mean SD of +/- 0.08 base pairs (bp) between runs, and single bp resolution of spiked TH01 allele 9.3 (198 bp) from TH01 allele 10 (199 bp) of the CTTv ladder with R = 0.81. A four-color multiplex sizing analysis of three different AmpFlSTR allelic ladders consisting of nine loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) and gender alleles (Amelogenin) on a 10-cm device had a mean SD of +/- 0.15 bp between runs for sizing three loci, i.e., FGA, D18S51 and D3S818; alleles differing by 2 bp in size were resolved with resolutions close to baseline. This work demonstrates that plastic microfluidic devices are capable of quality sequencing and STR sizing comparable to that of glass devices of similar separation lengths.

DNA, Single-Stranded↗
PubMed1994

Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes.

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 x 10(-6). Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-gamma genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR -gamma pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.

Adult↗
PubMed2001

A classification efficiency test of spectral karyotyping and multiplex fluorescence in situ hybridization: identification of chromosome homologies between Homo sapiens and Hylobates leucogenys.

Two digital fluorescence microscopy systems, spectral karyotyping (SKY) and multiplex fluorescence in situ hybridisation (M-FISH), are used with multicolour probe sets to assist in the detection of chromosome aberrations. We have compared the resolution of the two methods in their ability to identify karyotype rearrangements, which have occurred during the divergence of Homo sapiens and Hylobates leucogenys in evolution. A 24-color human paint kit distinguishes 74 conserved autosomal segments in H. leucogenys, some of which are difficult to resolve. We examined the extent to which the SKY and M-FISH techniques are able to detect the smallest of these bands. We have found this to be a rigorous test of multicolour chromosome classification systems. We conclude from our results that both systems are able invariably to classify the majority of conserved segments but differ in the efficiency of detection of small inserts.

Animals↗
PubMed2001

Karyotypic similarity identified by multiplex-FISH relates four prostate adenocarcinoma cell lines: PC-3, PPC-1, ALVA-31, and ALVA-41.

Recently developed molecular cytogenetic techniques for karyotyping are providing new and important insights regarding the chromosomal changes that occur in solid tumors. We used multiplex-FISH to analyze four adenocarcinoma cell lines, PC-3, PPC-1, ALVA-31, and ALVA-41, in which the characterization of a large number of rearranged chromosomes was partially or substantially inconclusive by G-banding. Although the original descriptions of these lines depict them as distinct entities established from different patients, this study demonstrates that these four lines share numerous, highly rearranged chromosomes, strongly supporting the conclusion that they are derived from the same patient material. Our analysis indicates that PPC-1, ALVA-31, and ALVA-41 were derived from PC-3 through mechanisms involving clonal progression represented by sequential changes and clonal diversion represented by differing patterns of changes. Extensive cellular heterogeneity was detected in all four lines, and most rearrangements included segments derived from multiple chromosomes. Each line also showed a set of unique derivative chromosomes. However, a limited number of metaphase cells (approximately 10) was analyzed for each line, and numerous single-cell abnormalities were detected in all of them. Therefore, it is plausible that the number of clonal, shared, and/or unique rearrangements has been underestimated. These cell lines have been utilized as models for understanding the biology of prostate cancer and reportedly differ in their cell physiology. Rather than detracting from their value, a complete understanding of the interrelationships of these lines to one another may provide the opportunity to define the molecular changes that have led to their individual malignant phenotypes.

Adenocarcinoma↗
PubMed1989

Characteristics of a multiplex IDDM sample: unexplained differences with other samples.

Characteristics of a multiplex sample of families with insulin-dependent diabetes mellitus (IDDM) are studied and contrasted with similar characteristics in other, more conventionally sampled data sets. Some characteristics remain consistent with earlier observations including the high frequency of human leukocyte antigen (HLA) DR3,4 in affected individuals and the greater than expected percentage of HLA haplotype sharing among affected sib pairs. In other respects, however, differences are seen between this sample and others. "Control" haplotypes, i.e., those not transmitted to the first affected offspring, had a higher frequency of DR3 and DR4 than expected, and a rather high frequency of affected parents was observed. Differences between the first affected and later affected offspring reported in other samples were absent from these families. No effect of the sampling scheme and the resulting distribution of parental phenotypes could be shown to explain this difference.

Child↗
PubMed1989

Genes predisposing to IDDM in multiplex families.

Analysis of HLA haplotypes occurring in more than one, only one, or no diabetics in GAW5 multiplex insulin-dependent diabetes mellitus (IDDM) families suggested: 1) DR3, DR4, DRw6, and DRw8 are positively associated, and DR2 is negatively associated, with IDDM; 2) DR4 haplotypes are more diabetogenic than DR3 haplotypes; some DR3 haplotypes lacking B8/B18 are more diabetogenic than those carrying B8/B18; DR3 haplotypes with DR beta TaqI bands [2,5,10/11] are more diabetogenic than those without; DR4 haplotypes that carry DQw3.2 are more diabetogenic than those that do not; some DR2 haplotypes are diabetogenic rather than protective. Analysis of DR3 and DR4 transmission from DR3/X and DR4wX (X not equal to 3,4) healthy parents suggested: 3) no distortion of transmission to healthy children and 4) mothers transmit DR4 (and perhaps DR3) less often than fathers to diabetic children. Analysis of INS, GM, and HLA haplotype sharing in affected sib pairs suggested: 5) random segregation of INS haplotypes and 6) a tendency to increased sharing of GM haplotypes in affected sib pairs who were HLA-identical and DR3/4, compared with pairs who were not.

Child↗
PubMed2003

Comparative multiplex dosage analysis detects whole exon deletions at the phenylalanine hydroxylase locus.

We have developed quantitative comparative multiplex dosage analysis to detect altered copy number of regions of the phenylalanine hydroxylase gene. Out of 41 alleles (4% of 1,010 PKU chromosomes) on which a mutation had not been characterized previously, this technique has highlighted two novel mutations: deletions of exon 5 and of exon 6 on a total of eight alleles. Restriction-enzyme digestion of genomic DNA and hybridization to an amplified segment of the phenylalanine hydroxylase (PAH) cDNA probe PAH247 established the size of the deletion in five individuals to be between 700 and 900 bases. We also report somatic mosaicism in the parent of an affected child previously shown to have a deletion spanning exons 5 and 6. Finally, we report a putative duplication of a region encompassing exon 6 in an affected individual.

Alleles↗