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PubMed2000

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays.

MOTIVATION: The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. RESULTS: To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.

Chromosomes, Artificial, Bacterial↗
PubMed2025

Tribus: semi-automated discovery of cell identities and phenotypes from multiplexed imaging and proteomic data.

MOTIVATION: Multiplexed imaging and single-cell analysis are increasingly applied to investigate the tissue spatial ecosystems in cancer and other complex diseases. Accurate single-cell phenotyping based on marker combinations is a critical but challenging task due to (i) low reproducibility across experiments with manual thresholding, and, (ii) labor-intensive ground-truth expert annotation required for learning-based methods. RESULTS: We developed Tribus, an interactive knowledge-based classifier for multiplexed images and proteomic datasets that avoids hard-set thresholds and manual labeling. We demonstrated that Tribus recovers fine-grained cell types, matching the gold standard annotations by human experts. Additionally, Tribus can target ambiguous populations and discover phenotypically distinct cell subtypes. Through benchmarking against three similar methods in four public datasets with ground truth labels, we show that Tribus outperforms other methods in accuracy and computational efficiency, reducing runtime by an order of magnitude. Finally, we demonstrate the performance of Tribus in rapid and precise cell phenotyping with two large in-house whole-slide imaging datasets. AVAILABILITY AND IMPLEMENTATION: Tribus is available at https://github.com/farkkilab/tribus as an open-source Python package.

Proteomics↗
PubMed1998

Array-based multiplex analysis of candidate genes reveals two independent and additive genetic risk factors for myocardial infarction in the Finnish population.

We analysed common variants of eight genes implicated previously as risk factors for coronary heart disease or myocardial infarction (MI) in a cross-sectional study on patients with a history of MI and in carefully matched controls from the Finnish population. The most common low density lipoprotein receptor mutations in Finland were also included in our analysis. Multiplex genotyping of the target genes was performed using a specific and efficient array-based minisequencing system. The 4G allele of the plasminogen activator inhibitor gene (P < 0.05) and the PlA2 allele of the glycoprotein IIIa gene (P < 0.01) were associated with an increased risk of MI in our study population. We analysed the combined effect of these risk alleles and found that the concurrent carrier status of the two genetic variants conferred a high risk for the development of MI in our sample (OR = 4.5, P = 0.001), which was particularly prominent in male subjects (OR = 6.4, P = 0.0005). This study demonstrates the application of a new powerful tool for genome analysis to yield information on the inherited determinants of susceptibility to MI. The observation of two separate genes contributing an additive risk of developing MI exemplifies the advantages of multiplex analysis of genetic variation.

Adult↗
PubMed2002

Multiplex interphase FISH as a screen for common aneuploidies in spontaneous abortions.

BACKGROUND: A multiplex fluorescence in-situ hybridization (FISH) strategy using chromosome-specific probes for eight chromosomes as an initial screen for chromosome abnormalities in uncultured tissues from spontaneous abortions was evaluated. METHODS: Fifty-seven prefetal spontaneous abortions were studied by karyotyping cultured cells and using FISH on uncultured cells. Two probe sets were used, identifying chromosomes 13, 15, 16, 18, 21, 22, X and Y. RESULTS: Abnormalities were detected in 53% of cases by karyotyping, and 54% of cases by FISH. FISH detected an abnormality in four of five cases where cultures failed, and in two cases where maternal cells apparently overgrew the culture. FISH missed four trisomies not identifiable with the probe sets, and one trisomy because one probe set was unscorable. FISH using these probes identified 83% of all abnormalities detected by karyotyping. CONCLUSIONS: FISH can detect abnormalities in a significant proportion of cases where the culture fails to grow or is contaminated by maternal cell growth. Multiplex FISH as an initial screen, followed by culture and karyotyping in cases where no abnormality is detected, would identify a higher proportion of chromosome abnormalities in spontaneous abortion specimens than karyotype analysis alone.

Abortion, Spontaneous↗
PubMed1999

Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects.

A great majority of patients seeking preimplantation genetic diagnosis (PGD) are women >35 years of age. In addition to being carriers for single gene defects, these women also have a higher risk of having children with Down's syndrome (trisomy 21). For these patients, it would be advantageous if a diagnostic test for trisomy 21 was developed, which could be used in conjunction with tests for single gene defects. Here, we assessed the feasibility of developing an accurate genetic test for diagnosing trisomy 21 and the mutation causing spinal muscular atrophy (SMA) in single cells using multiplex fluorescence polymerase chain reaction (PCR). Single- and two-round PCR were developed using a combination of primers for the survival motor neuron (SMN) gene exons 7 and 8 and two chromosome 21 short tandem repeats (STRs), D21S226 and D21S11. After only 36 cycles, 88 and 68% of normal single cells were screened for SMA mutations and trisomy 21 respectively. In multiplex PCR using only two primers (SMN exon 7 and D21S11) instead of four, the efficiency of SMA diagnosis was increased to 93%. In the same reactions, the D21S11 alleles were detected in 83% of the normal single cells. Clinical applications of this assay should enable detection of those embryos that have inherited three heterozygous alleles and, therefore, benefit many PGD patients who are at an increased risk of Down's syndrome.

Blastomeres↗
PubMed2001

Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination.

Large deletions in the dystrophin gene account for > 60% of mutations responsible for Duchenne muscular dystrophy (DMD). We have developed a genetic test that can be used directly for the preimplantation genetic diagnosis (PGD) of a majority of couples at risk of transmitting DMD. The test, a double nested multiplex polymerase chain reaction assay for the amplification of exons 8, 19, 45, 47 and 51 allows the detection of over 70% of all DMD deletions. Amelogenin sequences on the X and the Y chromosomes were also co-amplified to provide a correlation between embryo gender and deletion status. The setting up of reliable single cell assays for preimplantation genetic diagnosis is delicate and time consuming. Assays have to be validated on a large number of single cells for each specific mutation to assess efficiency and accuracy before being applied clinically. The multiplex procedure permitted the validation of all tested loci in the same series of isolated lymphocytes rather than in separate series for each exon. One hundred single lymphocytes, 50 female and 50 male cells, were analysed with an overall amplification rate of 98% and an amplification failure of 2% per exon. We suggest that this test is reliable, easy to set up and much preferable to a mere sex determination with the selective transfer of female embryos.

Dystrophin↗
PubMed1994

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

Autoanalysis↗
PubMed2002

Multiplex Pyrosequencing.

We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.

Animals↗
PubMed2002

Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry.

We report an approach using solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3'-biotinylated DNA products. These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to the MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS. We selected four biotin-ddNTPs with distinct molecular weights to generate extension products that have a 2-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum. Using this method, we simultaneously distinguished six nucleotide variations on synthetic DNA templates mimicking mutations in the p53 gene and two disease-associated SNPs in the human hereditary hemochromatosis gene.

Biotinylation↗
PubMed2003

A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed.

We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.

Animal Feed↗
PubMed2005

DNA analysis with multiplex microarray-enhanced PCR.

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3' end of a universal amplification primer. The complex primer pair is covalently tethered through its 5' end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 10(6)-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 10(3) genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.

DNA↗
PubMed2005

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.

Base Pair Mismatch↗
PubMed1997

Steatocystoma multiplex: a case with unusual clinical and histological manifestation.

A 35-year-old man at our clinic was found to have steatocystoma multiplex manifested by dome-shaped nodules confined to the scalp and forehead. One of four cyst biopsy specimens revealed large lining cells with granular cytoplasm adjacent to typical steatocystoma epithelial lining cells. Neither fibrosis nor inflammatory changes were noted in the surrounding connective tissue. The large granular lining cells had immunohistochemical characteristics of the macrophage/monocyte lineage. This case appears to be an unusual clinical and histological manifestation of steatocystoma multiplex.

Adult↗
PubMed2000

Pneumosinus dilatans multiplex, mental retardation, and facial deformity.

Pneumosinus dilatans is a term used to describe enlargement of one or more paranasal sinuses without radiological evidence of localized bone destruction, hyperostosis, or mucous-membrane thickening. To date, many cases have been reported that involved frontal, ethmoid, sphenoid, and maxillary sinus. However, no case has been reported that involved all paranasal sinuses. Our case involved mastoid air cells as well as all paranasal sinuses. It is named pneumosinus dilatans multiplex by us. This is the first case to be reported in English literature that has this syndromic condition of pneumosinus dilatans multiplex, mental retardation, and facial deformity.

Adult↗
PubMed1999

The etiology of genital ulcer disease by multiplex polymerase chain reaction and relationship to HIV infection among patients attending sexually transmitted disease clinics in Pune, India.

OBJECTIVES: To determine the etiology of genital ulcer disease (GUD) among patients attending sexually transmitted disease (STD) clinics in Pune, India, and to examine the relationship to HIV infection and compare the clinical diagnosis of GUD with the results of a multiplex polymerase chain reaction (M-PCR) assay for Treponema pallidum, herpes simplex virus (HSV), and Hemophilus ducreyi infection. METHODS: Between June 20, 1994, and September 26, 1994, 302 patients with a genital ulcer were evaluated. Clinical etiology of GUD was based on physical appearance and microbiologic evaluations which included darkfield microscopy and serology for syphilis. Swabs of each genital ulcer were tested for HSV antigen by enzyme immunoassay (Herpchek; Dupont, Wilmington, DE) and processed in a multiplex PCR assay (M-PCR; Roche, Branchburg, NJ) for simultaneous detection of HSV, Treponema pallidum, and Hemophilus ducreyi. RESULTS: Two hundred seventy-seven men and 25 women with a median age of 25 were evaluated. The seroprevalence of HIV was 22.2%. The etiology of GUD as determined by M-PCR was HSV (26%), H. ducreyi (23%), T. pallidum (10%), and multiple infections (7%); no etiology was identified in 34%. HIV seroprevalence was higher among those patients positive for HSV compared with other etiologies (OR = 2.1, CI: 1.2-3.7; p = 0.01). When compared with M-PCR, the Herpchek test was 68.5% sensitive and 99.5% specific. Darkfield detection for T. pallidum was 39% sensitive and 82% specific, in contrast to rapid plasma reagin and fluorescent treponemal antibody absorption test, which was 66% sensitive and 90% specific. Clinical diagnosis alone or in combination with basic laboratory tests showed poor agreement with M-PCR.

Adolescent↗
PubMed1997

Osteogenesis imperfecta with arthrogryposis multiplex congenita (Bruck syndrome)--evidence for possible autosomal recessive inheritance.

We report a son and a daughter of a first cousin Pakistani marriage who both have osteogenesis imperfecta and the son in addition has arthrogryposis multiplex congenita. Bruck [(1897): Dtsch Med Wochenschr 23: 152-155] first reported the case of a boy who had multiple fractures and joint ankylosis, subsequently only one sibship with three affected cases and seven sporadic cases have been reported to our knowledge. On the basis of consanguinity this suggests that the association of osteogenesis imperfecta and arthrogryposis multiplex congenita is inherited in this family as an autosomal recessive condition with variable expression.

Arthrogryposis↗
PubMed2000

A new syndrome of optic nerve colobomas and renal abnormalities associated with arthrogryposis multiplex.

Renal-coloboma syndrome is a developmental disorder involving optic nerve colobomas and renal hypoplasia/insufficiency, which exhibits autosomal dominant inheritance and a highly variable phenotype (OMIM:120330). Mutation in the PAX2 gene was found to result in the renal-coloboma phenotype. We report on an Arab family with autosomal dominant inheritance of a syndrome characterized by a variable combination of optic nerve colobomas, renal abnormalities, vesicoureteral reflux, lax joints and arthrogryposis multiplex. Apart from the arthrogryposis multiplex which has not been described in the renal-coloboma syndrome, the features of the syndrome in this family are very similar to the renal-coloboma syndrome. However sequencing of all 12 axons of PAX2 gene revealed no mutation in this family. The disorder in this family is likely to represent a new syndrome with features overlapping with the renal-coloboma syndrome.

Abnormalities, Multiple↗
PubMed2003

Multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts in paraffin-embedded tissues from patients with chronic myeloid leukemia and acute lymphoblastic leukemia.

Diagnosis of chronic myeloid leukemia and acute lymphoblastic leukemia requires the investigation of the Philadelphia chromosome translocation t(9;22) or the molecular detection of BCR-ABL fusion transcripts. Determination of the type of fusion transcript is crucial for quantitative molecular monitoring the course of the disease during treatment. Histopathologists, who usually use formalin-fixed tissues, may be confronted with the need to investigate the BCR-ABL rearrangement when evaluating tumor forming infiltrates and bone marrow trephines from patients presenting with chronic myeloproliferative disorders. Therefore, we have established a one-tube multiplex RT-PCR for the detection of common BCR-ABL fusion transcripts (b2a2, b3a2, e1a2) in routinely processed tissues and bone marrow trephines with respect to the inevitable fragmentation of ribonucleic acids in these specimens. RT-PCR products allow distinct and unequivocal differentiation of the underlying fusion in either the Major- or minor-breakpoint cluster region. Detection of BCR-ABL fusion transcripts by multiplex RT-PCR in routinely processed and fixed tissues is a time- and cost-sparing tool for definite diagnosis of typical chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.

Bone Marrow Cells↗