PCR detection of bacteria in seven minutes.
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Bacteria↗
Biomedical subjects
P Belgrader
Publications and source records attributed to P Belgrader.
Analysis of DNA fragments from conventional and microfabricated PCR devices using delayed extraction MALDI-TOF mass spectrometry.
Applications of polymerase chain reaction (PCR) product analysis using rapid affinity capture followed by delayed extraction (DE) MALDI-TOFMS is presented. Such applications include multiplex short tandem repeat (STR) typing, which is demonstrated for STR systems from conventional and microchip-based thermal cycling instruments. Using the combination of the microfabricated PCR instrument and DE-MALDI-TOFMS, a complete genotyping assay can be performed in under 50 min with a resultant molecular weight accuracy approaching or exceeding 100 ppm through external calibration. The observed resolution and mass accuracy for a 69-base PCR product enables identification of single base substitutions by direct molecular weight determination.
Calibration↗
Real-time microchip PCR for detecting single-base differences in viral and human DNA.
This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.
DNA↗
Rapid PCR for identity testing using a battery-powered miniature thermal cycler.
A microfabricated, battery-powered thermal cycler was implemented in PCR-based DNA typing for human identification. HLA DQ alpha and an STR triplex were PCR amplified using a device known as the Miniature Analytical Thermal Cycling Instrument (MATCI). The extremely efficient heating properties of the MATCI enabled thermal cycling to be completed in as little as 21 min. In addition, the feasibility of using the real-time fluorescent detection system of the MATCI was demonstrated. The successful application of this portable, prototype device to forensic identity testing is a significant milestone towards the eventual development of a completely integrated DNA testing instrument that would also incorporate sample preparation and allele detection.
DNA↗
Rapid pathogen detection using a microchip PCR array instrument.
An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 10(5)-10(7) organisms/L (10(2)-10(4) organisms/mL).
Bacillus subtilis↗
DNA typing of human leukocyte antigen sequence polymorphisms by peptide nucleic acid probes and MALDI-TOF mass spectrometry.
A novel analytical method using PNA probes detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to type sequence polymorphisms within the human leukocyte antigen (HLA), DQA locus. Streptavidin-coated magnetic beads were used to immobilize biotinylated DNA. PNA probes representing possible alleles were then prepared for the immobilized DNA hybridization. The nonspecific PNA probes were removed with stringent washes. The PNA/DNA/beads conjugate was analyzed by MALDI-TOFMS. The genotype of the DNA was determined by the detected molecular masses of the released PNA probes. Reproducible and accurate genotyping was achieved by this analytical method.
DNA↗
Discrimination of single-nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry.
Human genomic and mitochondrial DNA contain large numbers of single-nucleotide polymorphisms (SNPs), many of which are linked to known diseases. Rapid and accurate genetic screening for important SNPs requires a general methodology which is easily implemented. We present here an approach to SNP discrimination based on high-specificity hybridization of peptide nucleic acid (PNA) probes to PCR-amplified DNA. The assay is directly applied to polymorphisms located within hypervariable region 1 of the human mitochondrial genome and type 1 suballeles of the human leukocyte antigen DQ alpha gene. Captured, single-stranded DNA molecules prepared by PCR amplification are hybridized with PNA probes in an allele-specific fashion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is then used for rapid, precise, and unambiguous detection and identification of the hybridized PNA probes. Since PNA oligomers bind strongly to complementary DNA under minimal salt conditions, the use of PNA probes is compatible with MALDI-TOFMS. The unparalleled ability of MALDI-TOFMS analysis in terms of molecular weight resolution and accuracy, in conjunction with the highly specific PNA hybridization afforded by this method, offers promise for development into a multiplexed, high-throughput screening technique.
DNA↗
Analysis of short tandem repeat polymorphisms in human DNA by matrix-assisted laser desorption/ionization mass spectrometry.
The analysis of an important class of human genetic polymorphisms, short tandem repeats (STRs), using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. Several model STR systems have been investigated to evaluate MALDI-TOFMS as a realistic alternative to established electrophoresis procedures, and to develop rapid and generally applicable approaches to polymerase chain reaction (PCR) product purification for MALDI-TOFMS analysis. A purification/preconcentration method for PCR product preparation based on affinity capture of biotin-labeled PCR products is demonstrated to be directly compatible with MALDI-TOFMS analysis. The entire sample preparation for MALDI-TOFMS analysis immediately following PCR amplification from human DNA extracts can be accomplished routinely in under 12 min in a single Eppendorf tube. The simplicity of this approach essentially eliminates the sample preparation bottleneck encountered with MALDI-TOFMS analysis of PCR products using existing methods. Using this method, encouraging genotyping results are demonstrated for the THO1 and TPOxx STR systems using subpicomole quantities that represent a fraction of the original dsDNA from a single PCR reaction. The technique is also demonstrated to facilitate rapid sizing of PCR fragments larger than 200 bases using MALDI-TOFMS. As described here, the analysis of DNA can be accomplished in a manner that takes advantage of the rapid and accurate analysis capabilities offered by MALDI-TOFMS.
DNA↗
DNA typing in thirty seconds with a microfabricated device.
We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microfluidic electrophoresis device. We have achieved baseline-resolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci CSF1PO, TPOX, THO1, and vWA (abbreviated as CTTv) were performed in less than 2 min. This constitutes a 10- to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 micron x 100 micron in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50 degrees C. A fluorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 microl was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.
DNA↗
Peptide nucleic acid characterization by MALDI-TOF mass spectrometry.
Peptide nucleic acids (PNAs) are a new class of DNA mimics in which the regular nucleobases of adenine, thymine, cytosine, and guanine are connected via a peptide-like backbone. PNA molecules retain the same Watson-Crick base pairing as regular oligonucleotides, with the added benefits of greater specificity and resistance to enzymatic digestion. While the use of PNAs has grown rapidly because of their potential applications in biotechnology, little work has been done on developing analytical procedures for characterizing them. We have found matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to be an effective tool for PNA analysis. PNA molecules survive the MALDI process intact and are easily ionized with almost no multiply-charged species. These features allow mixtures to be easily characterized. Traditional protein matrices (e.g., sinapinic acid,2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid) were found to be superior to DNA matrices (e.g., trihydroxy-acetophenone, 3-hydroxypicolinic acid, picolinic acid). In addition, the new DNA matrix 6-aza-2-thiothymine worked well. The ability of MALDI-TOF-MS to ascertain PNA purity and sequence information at low picomole levels will be important as greater quality control of PNA synthesis is needed (e.g., when PNAs are used as antisense or antigene drugs).
Base Sequence↗
Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis.
The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.
Automation↗
Characterization of mitochondrial DNA using low-stringency single specific primer amplification analyzed by laser induced fluorescence--capillary electrophoresis.
Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP-PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D-loop region is described. The DNA from five individuals were amplified (first step) using sequence-specific primers to produce 1021 bp fragments containing the D-loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO-1 and laser-induced fluorescence (LIF) detection. The LSSP-PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.
DNA Primers↗
Reusing the same blood-stained punch for sequential DNA amplifications and typing.
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Blood Preservation↗
PCR-based human leukocyte antigen (HLA) DQ alpha typing of blood stained light and dark blue denim fabric.
Obtaining typable PCR products from DNA purified from blood stained blue denim has been difficult since inhibitors of PCR in blue denim apparently co-purify with the DNA. Organic and chelex extraction methods were tested for their ability to purify typable DNA from either light or dark blue denim fabric both stained with blood. DNA purified from the light blue denim using either method was successfully used in obtaining correct HLA-DQ alpha typing results. The chelex, but not the organic, procedure was able to yield typable DNA when the dark blue denim was the substrate. Therefore, the chelex method may be more effective than the organic method in preventing compounds that inhibit PCR from co-purifying with the DNA.
Blood Stains↗
The evaluation of fast purification methods for preparing polymerase chain reaction (PCR) products for capillary electrophoresis analysis.
The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column, hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing, and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time, and cost.
DNA↗
Automated DNA purification and amplification from blood-stained cards using a robotic workstation.
A new method for the purification of DNA on blood-stained cards was developed. This method was implemented into a high-throughput automated system using a Biomek 1000 robotic workstation. In addition, the processes of DNA purification and amplification were coupled into a completely automated and uninterrupted prototype system, and the resultant PCR products generated by this system were subjected to automated desalting for capillary electrophoresis analysis.
Autoanalysis↗
Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life.
Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.
Animals↗
Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance.
The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations of the type that cause translation to terminate prematurely at or upstream of codon 189 within exon 6 reduce the level of nuclear TPI mRNA to 20 to 30% of normal by a mechanism that is not a function of the distance of the nonsense codon from either the translation initiation or termination codon. In contrast, frameshift and nonsense mutations of another type that cause translation to terminate prematurely at or downstream of codon 208, also within exon 6, have no effect on the level of nuclear TPI mRNA. In this work, quantitations of RNA that derived from TPI alleles in which nonsense codons had been generated between codons 189 and 208 revealed that the boundary between the two types of nonsense codons resides between codons 192 and 195. The analysis of TPI gene insertions and deletions indicated that the positional feature differentiating the two types of nonsense codons is the distance of the nonsense codon upstream of intron 6. For example, the movement of intron 6 to a position downstream of its normal location resulted in a concomitant downstream movement of the boundary between the two types of nonsense codons. The analysis of intron 6 mutations indicated that the intron 6 effect is stipulated by the 88 nucleotides residing between the 5' and 3' splice sites. Since the deletion of intron 6 resulted in only partial abrogation of the nonsense codon-mediated reduction in the level of TPI mRNA, other sequences within TPI pre-mRNA must function in the effect. One of these sequences may be intron 2, since the deletion of intron 2 also resulted in partial abrogation of the effect. In experiments that switched introns 2 and 6, the replacement of intron 6 with intron 2 was of no consequence to the effect of a nonsense codon within either exon 1 or exon 6. In contrast, the replacement of intron 2 with intron 6 was inconsequential to the effect of a nonsense codon in exon 6 but resulted in partial abrogation of a nonsense codon in exon 1.
Amino Acid Sequence↗