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PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2002

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations.

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

Alleles↗
PubMed2003

Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry.

Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites.

Base Sequence↗
PubMed2003

Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities.

We have developed a novel, spectroscopic technique for high-sensitivity, label-free DNA quantification. We demonstrate that an optical resonance (whispering gallery mode) excited in a micron-sized silica sphere can be used to detect and measure nucleic acids. The surface of the silica sphere is chemically modified with oligonucleotides. We show that hybridization to the target DNA leads to a red shift of the optical resonance wavelength. The sensitivity of this resonant technique is measured as 6 pg/mm(2) mass loading, higher as compared to most optical single-pass devices such as surface plasmon resonance biosensors. Furthermore, we show that each microsphere can be identified by its unique resonance wavelength. Specific, multiplexed DNA detection is demonstrated by using two microspheres. The multiplexed signal from two microspheres allows us to discriminate a single nucleotide mismatch in an 11-mer oligonucleotide with a high signal-to-noise ratio of 54. This all-photonic whispering gallery mode biosensor can be integrated on a semiconductor chip that makes it an easy to manufacture, analytic component for a portable, robust lab-on-a-chip device.

Base Pair Mismatch↗
PubMed2003

Multiplexed-replica exchange molecular dynamics method for protein folding simulation.

Simulating protein folding thermodynamics starting purely from a protein sequence is a grand challenge of computational biology. Here, we present an algorithm to calculate a canonical distribution from molecular dynamics simulation of protein folding. This algorithm is based on the replica exchange method where the kinetic trapping problem is overcome by exchanging noninteracting replicas simulated at different temperatures. Our algorithm uses multiplexed-replicas with a number of independent molecular dynamics runs at each temperature. Exchanges of configurations between these multiplexed-replicas are also tried, rendering the algorithm applicable to large-scale distributed computing (i.e., highly heterogeneous parallel computers with processors having different computational power). We demonstrate the enhanced sampling of this algorithm by simulating the folding thermodynamics of a 23 amino acid miniprotein. We show that better convergence is achieved compared to constant temperature molecular dynamics simulation, with an efficient scaling to large number of computer processors. Indeed, this enhanced sampling results in (to our knowledge) the first example of a replica exchange algorithm that samples a folded structure starting from a completely unfolded state.

Algorithms↗
PubMed2003

A simple method for the detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase chain reaction.

BACKGROUND: Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles. METHOD: Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results. RESULTS: We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence. CONCLUSION: This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.

Alleles↗
PubMed2000

A method for homogeneous color-compensated genotyping of factor V (G1691A) and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR.

OBJECTIVES: To set up an optimized multiplex polymerase chain reaction for real-time genotyping of the prothrombotic risk factors methylenetetrahydrofolate reductase C677T and factor V Leiden on the LightCycler. DESIGN AND METHODS: Novel primer and probe sets were designed on the basis of thermodynamic double-strand DNA stability calculations. Detection probes were labeled with LC-Red640 or Cy5.5 dye. RESULTS: The polymerase chain reaction efficiency was reduced in multiplex polymerase chain reaction but this could be overcome by the design of novel amplification primers. The selection of detection probes with a lower melting temperature (T(m)) and high Delta T(m) improved the discrimination of heterozygous samples. Color compensation was not compromised by either the use of the Cy5.5 dye or different fluorescein linker chemistries. CONCLUSIONS: Probes with a Delta T(m) of 5 degrees C or more between the matched and mismatched state are desirably for genotyping. Such probes can be selected by using a priori calculations based on the thermodynamic nearest neighbor model.

DNA Mutational Analysis↗
PubMed1990

Continuous monitoring of a changing sample by multiplex gas chromatography.

Multiplex gas chromatography (MGC) is a technique in which multiple samples may be introduced into a chromatographic system regardless of the elution time of the individual components. Although the output obtained from a MGC experiment is not directly interpretable, computational techniques can be used to obtain the chromatogram from the detector output data. This is done by calculating the impulse response function from the multiplexed output data.

Butanes↗
PubMed2000

Multiplexed particle-based flow cytometric assays.

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, 'no wash' assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.

Cytokines↗
PubMed2002

Simultaneous genotyping of single nucleotide polymorphisms in the IL-1 gene complex by multiplex polymerase chain reaction-restriction fragment length polymorphism.

The interleukin-1 (IL-1) gene complex consists of the IL-1alpha, IL-1beta and IL-1 receptor antagonist genes. Single-nucleotide polymorphisms (SNP) in all three genes have been associated with human diseases. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a restriction site for endonuclease AlwNI or XcmI in the presence of allele-specific nucleotides at the polymorphic positions. Based on this technique, a simple and robust multiplex polymerase chain reaction/restriction fragment length polymorphism (multiplex PCR/RFLP) assay was developed to determine simultaneously three to four informative SNPs (IL-1beta/+3954, IL-1beta/-511 and IL-1Ra/9261 or IL-1alpha/-889, IL-1beta/-31, IL-1beta/5810 and IL-1Ra/11100 SNPs) in the IL-1 gene complex.

Alleles↗
PubMed2003

Semiquantitative and qualitative assessment of B-lymphocyte V H repertoire by a fluorescent multiplex PCR.

We established a new tool to perform semiquantitative and qualitative screening for V(H) gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with V(H) family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular V(H) family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative V(H) family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of V(H) family usage and an immediate determination of the involved V(H) gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for V(H) repertoire screening of larger populations of patients suffering from diseases with changes in the V(H) repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.

Adult↗
PubMed1999

Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay.

Several methods have been developed to quantify cytokines and chemokines in biological fluids and tissue culture samples, including bioassays, enzyme-linked immunosorbent assay (ELISA), intracellular staining, ribonuclease protection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we describe a new multiplexed assay, using the FlowMetrix system, that can quantify multiple cytokines simultaneously in a small sample volume. This assay was found to be more accurate, sensitive and reproducible than the conventional microtitre ELISA procedure. Furthermore, the time and cost involved are comparable to, or less than, the ELISA. A key feature of the FlowMetrix assay is its ability to multiplex: here, we show that this assay can accurately quantitate 15 cytokines in a 100 microl sample volume while the same analysis by ELISA requires 1.5 ml (100 microl for each cytokine assay). By using this Flow Metrix assay, we could demonstrate that only T helper 1 (T(H)1)-deviated cells produce detectable levels of interleukin (IL)-2, while only T(H)2-deviated cells produce significant amounts of IL-4. Six other cytokines were produced by both T cell subsets, with the T(H)1 population producing more IL-3, granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon (IFN)-gamma, and the T(H)2 population producing more IL-5, IL-10, and IL-13. Seven other cytokines were not produced in detectable amounts. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response.

Animals↗
PubMed1981

Arthrogryposis multiplex congenita: an autopsy case of a fatal form.

A case of a severe and fatal form of arthrogryposis multiplex congenita with a full necropsy examination is presented in which the central nervous system and many muscles of the four extremities were examined histologically. The most striking feature was a great reduction in the muscular tissue of the limbs with a marked increase in the adipose tissue. The muscular changes were thought to be caused by neurogenic atrophy, and microscopy of the spinal cord revealed developmental abnormalities, including degenerative changes of the anterior horn cells. Neonatal and fatal cases of arthrogryposis multiplex congenita reported in the literature are briefly reviewed, and the characteristics of the present case and its relation to other congenital neuromuscular disorders are discussed.

Adipose Tissue↗
PubMed1998

Detection and clearance of prostate cells subsequent to ultrasound-guided needle biopsy as determined by multiplex nested reverse transcription polymerase chain reaction assay.

OBJECTIVES: To determine if circulating prostate cells are detectable subsequent to transrectal ultrasound (TRUS)-guided biopsy, and if so, whether cells remain in circulation for up to 4 weeks. METHODS: Blood samples were drawn from 90 patients with elevated serum prostate-specific antigen (PSA) levels and/or abnormal digital rectal examination. Two samples were drawn from all patients immediately prior to TRUS and 30 minutes postbiopsy. Blood samples were also obtained 1 week postbiopsy from 83 patients, and 1 month postbiopsy from 61 patients. Multiplex nested reverse transcription polymerase chain reaction assay (RT-PCR) for PSA and prostate-specific membrane antigen (PSM) was performed on total ribonucleic acid (RNA) from each sample. Results were reported as positive if at least one of the targets was detected. RESULTS: Of 45 patients with biopsy-proven adenocarcinoma, 22 were RT-PCR positive prebiopsy and all remained positive 30 minutes postbiopsy. Of 23 patients with adenocarcinoma who were RT-PCR negative prebiopsy, 5 (22%) converted to positive 30 minutes postbiopsy (P < 0.001). Four of these 5 patients returned to negative after 1 week or 1 month. Of 45 patients without cancer at biopsy, 32 were RT-PCR negative prebiopsy and 6 (19%) converted to positive 30 minutes postbiopsy (P < 0.001). Although four of six available samples were still positive at 1 week, all four samples available 1 month postbiopsy were negative. CONCLUSIONS: Detection of circulating prostate cells subsequent to biopsy occurred in 11 of 55 (20%) previously RT-PCR negative patients, a proportion twice that reported in the literature. We attribute this higher proportion to the simultaneous detection of PSA and PSM mRNA in our multiplex assay. Conversion rates were similar in patients regardless of biopsy result. Testing of serial postbiopsy blood demonstrates clearing of these cells by 4 weeks in most patients.

Aged↗
PubMed2001

Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.

The objective of this study was to establish a rapid and reliable PCR method for the sexing of 8- to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male- and bovine-specific DNA, respectively. But the unequal number of copies of these two repetitive sequences required some modification of the multiplex PCR method. In consecutive and multiplex PCR, the first 10 PCR cycles were done with male-specific primer followed by an additional 23 cycles with bovine-specific primer. In this PCR method, the appearance of male- and bovine-specific bands was independent of the DNA concentration. This PCR method was applied successfully using groups of 8, 4, 2, and 1 blastomeres dissociated from the embryos, and the sexing efficiency was 100.0, 96.3, 94.3 and 92.1%, respectively. The coincident rate of sex determination between biopsied single blastomere and matched blastocyst was 90.0%. Therefore the developmental potential from 8- to 16-cell stage embryos to the blastocyst stage was not significantly different (P>0.2) for intact embryo (42.3%) than for demi-embryos (53.8%), suggesting that trauma to the demi-embryo caused by single-blastomere aspiration using a bevelled micropipette was very small. In conclusion, we developed a rapid (within 2 hours) and effective PCR method for the sexing of 8- to 16-cell stage bovine embryos using a single blastomere.

Animals↗
PubMed1999

Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction.

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos.

Animals↗
PubMed2003

Obsessive-compulsive behaviors in parents of multiplex autism families.

Parents of autistic probands with high and low rates of repetitive behaviors were compared for rates of obsessive-compulsive traits and disorder. The rate of repetitive behaviors was assessed using the Autism Diagnostic Interview-Revised (ADI-R) in 176 autistic probands from 57 multiplex families. Obsessive-compulsive disorder (OCD) in parents was determined by direct interview using a parental history questionnaire, with screening for obsessive-compulsive traits using the Yale-Brown Obsessive-Compulsive Scale checklist. Children who had high total scores on the repetitive behavior domain of the ADI-R were significantly more likely to have one or both parents with obsessive-compulsive traits or disorder compared with children who had low total scores on this domain. Children with high scores on D1/D2 of the ADI-R (narrow restricted interests and rituals) were significantly more likely to have one or both parents with OCD, especially fathers, than those with low D1/D2. The occurrence of obsessive-compulsive traits or disorder in parents of autistic children in multiplex families is significantly more likely if autistic children have a high occurrence of repetitive behaviors. Dichotomizing autistic probands by severity and type of repetitive behaviors (circumscribed interests and compulsive rituals) may yield more homogenous groups, which could be helpful in genetic linkage studies.

Adult↗
PubMed2000

Rapid molecular epidemiologic studies of human parainfluenza viruses based on direct sequencing of amplified DNA from a multiplex RT-PCR assay.

Sequencing studies of limited regions of the human parainfluenza viruses (HPIVs) genomes have helped describe patterns of virus circulation and characterize institutional outbreaks of HPIVs-associated respiratory illness. In this study, we sequenced reverse transcription polymerase chain reaction (RT-PCR)-amplified HPIVs RNA obtained from a multiplex RT-PCR assay described previously for simultaneous detection of HPIV-1, 2 and 3. Differences in the nucleotide sequences of limited regions of the HN gene allowed us to distinguish temporally and geographically diverse HPIV isolates (43 HPIV-1, 7 HPIV-2, 12 HPIV-3 isolates from this and previously published studies). In addition, an outbreak of HPIV-3-associated illness among infants on a pediatric ward was investigated by comparing sequences of three ward isolates with three matched community controls. Sequences of all ward isolates were identical and differed from those of the community controls, suggesting a single introduction and nosocomial transmission of the virus. Combining multiplex reverse transcription polymerase chain reaction (RT-PCR) assays with direct sequencing of the PCR products can provide an integrated system for rapid diagnosis and characterization of HPIVs.

Child↗