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PubMed1995

Fluorescence energy-transfer cyanine heterodimers with high affinity for double-stranded DNA. II. Applications to multiplex restriction fragment sizing.

Energy-transfer cyanine dyes, a thiazsole orange-thiazole-indolenine (butylTOTIN) and a thiazole orange-thiazole blue heterodimer (pentylTOTAB), form high-affinity complexes with double-stranded (ds)DNA with donor fluorescence (lambdaF(max)527 nm) quenched >90% and with acceptor fluorescence emission above 650 nm (S. C. Benson, Z. Zeng, and A. N. Glaser (1995) Anal. Biochem. 231, 247-255). After separation by agarose gel electrophoresis, bands of precomplexed dsDNA-dye restriction fragments containing 10 pg of dsDNA are readily detected with a laser-excited confocal-fluorescence gel scanner (R. A. Mathies et al. (1994) Rev. Sci. Instrum. 65, 807-812) following donor excitation at 488 nm (argon ion laser) or direct acceptor excitation at 647 nm (krypton ion laser). Accurate two-color multiplex sizing of restriction fragments is obtained with 488-nm excitation with dsDNA fragments precomplexed with thiazole orange dimer (TOTO) as unknowns (detected at 500-565 nm, green channel) and dsDNA fragments stained with the energy-transfer cyanine dyes (detected at 645-750 nm, red channel) as internal standards. There is negligible cross talk of fluorescence between the red and green channels and no significant dye migration in mixtures of prelabeled standard and unknown fragments.

DNA↗

PubMed1998

Multiplex display polymerase chain reaction amplifies and resolves related sequences sharing a single moderately conserved domain.

We present a technique, multiplex display polymerase chain reaction (MD-PCR), that amplifies and resolves coding sequences from messenger RNAs sharing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows detection of known and novel genes in a family by using one degenerate primer complementary to a gene family-specific domain. A second common primer is complementary to an oligonucleotide ligated to a nearby restriction enzyme cleavage site. Uniquely, restriction enzyme digestion of single-stranded cDNA, a technique never previously performed to useful advantage, is used to increase the specificity and sensitivity of the technique. Up to several hundred bases of coding sequence are amplified simultaneously from many (potentially from all) genes in a specific family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. Typically, more than 50% of the amplified sequences are from the targeted gene family and many of the amplified products are novel sequences. mRNAs representing less than 1 in 100,000 messages can be detected. The method allows the focused yet open-ended examination of genes in families known to be important in both normal cellular homeostasis and the etiology of many diseases.

Animals↗

PubMed1999

Two-color multiplexed analysis of eight short tandem repeat loci with an electrophoretic microdevice.

Single-channel microfabricated electrophoretic devices equipped with a dual-wavelength laser-induced fluorescence detection system were used for the fast analysis of an eight-loci, two-color multiplex short tandem repeat (STR) system for human identification. Routine analyses of the eight loci (CSF1PO, TPOX, TH01, vWA and D16S539, D7S820, D13S317, D5S818), requiring four-base resolution, were performed in only 2 min. Specific analyses for a microvariant allele (allele 9.3 of the TH01 locus) demanded single-base resolution and was performed in less than 10 min. The high accuracy of the microdevice for real-world STR sample analyses was demonstrated by comparison with conventional slab-gel electrophoresis. Our results show that a fast multiwavelength multichannel electrophoretic microsystem will be capable of routinely processing thousands of complex STR samples per day.

Color↗

PubMed1999

Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.

Arylamine N-Acetyltransferase↗

PubMed2001

A new type of phosphorescent nanospheres for use in advanced time-resolved multiplexed bioassays.

A new concept to design phosphorescent nanospheres is presented. The spheres are distinguishable by their individual decay time and spectral distribution of their emission spectra. They are composed of a phosphorescent ruthenium metal-ligand complex (MLC) dissolved, along with certain strongly fluorescent cyanine dyes, in modified polyacrylonitrile-based nanospheres. Since the emission spectrum of the MLC overlaps the absorption spectrum of the cyanine and both the MLC (the donor) and the cyanine (the acceptor) are in close spatial proximity, efficient resonance energy transfer (RET) does occur. Thus, the nanospheres emit dual luminescence, one from the acceptor dye and the other from the donor MLC. Variation of the concentrations of the acceptor dye results in a varying efficiency of RET, thus making the spheres distinguishable. Hence, a set of multiplexable sphere labels is obtained by using one MLC (acting as the phosphorescent donor and present in constant concentration) and one acceptor dye (which varies in terms of both spectral properties and concentration). The nanospheres can be identified by the emission maximum (reflecting the kind of acceptor dye) and by decay time (reflecting its concentration). Since the same donor MLC is used throughout, all nanospheres can be excited with the same light source.

Biological Assay↗

PubMed2001

Interleukin 1 gene cluster polymorphisms in multiplex families with spondylarthropathies.

Interleukin 1alpha (IL-1alpha) has been implicated in the pathogenesis of infectious, auto-immune and inflammatory diseases. Polymorphisms in the genes encoding IL-1alpha, IL-1beta and the IL-1 receptor antagonist (ILRN) molecules have been associated with several inflammatory diseases. As the marker D2S160 has been characterized as a candidate locus for Ankylosing Spondylitis (AS) after a genome-wide scan, and since this locus is located approximately 0.3 cM telomeric to the IL-1 gene cluster, we hypothesized that these cytokines might be good candidates for Spondylarthropathies (SpA). Therefore we tested this hypothesis using the transmission disequilibrium test (TDT) in 37 families representing 217 subjects from three intragenic IL-1 gene cluster polymorphisms, the IL1B Taq I, the ILRN 2nd intron, and the IL1A Nco I, taking HLA B27 status into account or not. In conclusion, by means of intra-familial TDT analysis we found no linkage or intra-familial association between SpA and the three IL-1 gene-cluster polymorphisms in SpA multiplex family material.

Adult↗

PubMed1995

Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction.

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems.

Adrenal Hyperplasia, Congenital↗

PubMed1996

Rapid detection of mitochondrial sequence polymorphisms using multiplex solid-phase fluorescent minisequencing.

This work describes a novel method, multiplex solid-phase fluorescent minisequencing, for the simultaneous detection of several point mutations and/or small deletions and insertions. The method is applied to the analysis of mitochondrial DNA polymorphisms for the purposes of individual identification. A database of 152 British Caucasians and 103 British Afro-Caribbeans has been constructed, and the probability of a chance match between two unrelated individuals is calculated as 0.054 for Caucasians and 0.026 for Afro-Caribbeans.

Base Sequence↗

PubMed1998

An anchored framework BAC map of mouse chromosome 11 assembled using multiplex oligonucleotide hybridization.

Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an "overgo" computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.

Animals↗

PubMed2001

Simultaneous identification of mutations by dual-parameter multiplex hybridization in peptide nucleic acid-containing virtual arrays.

The physical entrapment of peptide nucleic acids (PNA) in electrophoresis media provides a system for performing real-time hybridization. DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands. A second parameter, that of amplicon length, has been introduced to perform multiplex analyses on several mutations simultaneously. Size fractionation creates a virtual array of PCR products that can hybridize to one of a set of mutation-specific PNAs present within the matrix. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is characterized by its interaction with a specific PNA that gives a visually resolved distinction between wildtype and mutant allele. In contrast to conventional hybridization, heterozygotes are readily distinguished from homozygotes. Using a capillary electrophoresis-based DNA sequencer, this approach has been used to automate the identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene.

Base Sequence↗

PubMed2002

Multiplex analysis of single-nucleotide extension products on a 16-capillary, denaturing, high-performance liquid chromatography array.

We constructed a high-performance liquid chromatography array consisting of 16 monolithic poly(styrene/divinylbenzene) capillaries for the parallel multiplex analysis of fluorescent dye-labeled single-nucleotide extension products. Because of the high chemical and physical robustness of the column bed that is covalently linked to the inner surface of the fused silica capillary, the array can be reused thousands of times without replenishment. The choice of fluorophore exerts a significant effect on resolution of the extension products. FAM, HEX, and TAMRA allowed complete resolution of all four possible allelic extension products not only from the extension primer but also from each other. The quantitative accuracy of the method enables the genetic typing of bi- and triallelic single-nucleotide polymorphisms in polyploid genomes and pooled samples.

Chromatography, High Pressure Liquid↗

PubMed1995

Simultaneous detection by consensus multiplex PCR of high- and low-risk and other types of human papilloma virus in clinical samples.

A consensus multiplex PCR (CM-PCR) technique was developed to detect high-risk (HPV 16/18), low-risk (HPV 6/11), and over 40 other types of human papillomavirus (HPV), separately but simultaneously, by mixing three pairs of consensus primers in the same PCR mixture, for gene amplification. Simultaneous detection of three groups of HPV DNA provides valuable information for clinical practice and this procedure is simple and convenient for routine laboratory examinations. We detected HPV DNA sequences in plasmid HPV DNA and DNA extracted from tissues of condyloma acuminata and cervical carcinoma and from exfoliated cells of the lower genital tract of healthy Chinese women living in the People's Republic of China. We confirmed that this simple, convenient, and cost-beneficial CM-PCR technique is reliable for the detection of HPV DNA sequences.

Adolescent↗

PubMed1995

Non-linkage of a T-cell receptor gamma chain microsatellite (D7S485) to rheumatoid arthritis in multiplex families.

A highly informative microsatellite marker, D7S485, from the T-cell receptor gamma (TCRG) locus, has been used to study segregation of TCRG genes in 26 multiplex rheumatoid arthritis (RA) families. We used the sib-pair method to assess excess identity-by-descent sharing among affected members in these families and the LINKAGE package of programs was used to calculate two-point lod scores for the D7S485 marker. There was no evidence for segregation of TCRG genes with RA in affected siblings and significantly negative lod scores were obtained from linkage analyses using both autosomal dominant and recessive models of inheritance.

Adolescent↗

PubMed1999

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.

Alleles↗

PubMed1995

A fluorescent multiplex ARMS method for rapid mutation analysis.

The amplification refractory mutation system (ARMS) is a powerful technique for the identification of mutations. We present a modification of this technique involving coamplification of normal and mutant alleles with primers that differ in length by three to five bases. Fluorescent labelling allows exact sizing of the amplification products on an automated DNA fragment analyser. We have used the fluorescent multiplex ARMS method for the identification of common phenylketonuria mutations in Northern Ireland (R408W, 165T and F39L) together with the analysis of a polymorphic short tandem repeat site at the human phenylalanine hydroxylase locus. This provides an efficient and inexpensive first step for diagnostic mutation analysis in our population.

Alleles↗

PubMed1996

Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR.

The presence or absence of the mecA gene, the determinant of resistance to all beta-lactam antibiotics, was examined in clinical isolates of Staphylococcus aureus by multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bp nuc- based PCR fragment (amplification product of the nuc gene encoding specific Staphylococcus aureus nuclease) and a 533-bp mecA-based PCR fragment (amplification product of the mecA gene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.

Genes, Bacterial↗

PubMed1997

Distribution of dystrophin gene deletions mapped by multiplex PCR in the Moravian population.

Twenty two Duchenne muscular dystrophy (DMD) patients from the province of Moravia, Czech Republic, were tested for the presence of dystrophin gene rearrangements using multiplex polymerase chain reaction (PCR). Using primer pairs for amplification of two promoter regions and 27 exons, 11 patients were found positive for deletions spanning one or more exons. In all these cases, the deletions affected the distal part of the dystrophin gene, beginning from exon 44 but not reaching exon 60.

Cytoskeletal Proteins↗

PubMed1997

Multiplex PCR for avian pathogenic mycoplasmas.

Mycoplasma infections are of great concern in avian medicine, because they cause economic losses in commercial poultry production. A multiplex polymerase chain reaction (PCR) was optimized to simultaneously detect four pathogenic species of avian mycoplasmas. Four sets of oligonucleotide primers specific for Mycoplasma gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI) were used in the test. By using agarose gel electrophoreses for detection of the PCR-amplified DNA products, the sensitivity of detection was between 1 pg for MG, 1 pg for MS, 100 fg for MM and 100 pg for MI after 35 cycles of PCR. Similar sensitivity of these primers was achieved with broth cultures of these four organisms.

Animals↗