[Show abstract][Hide abstract] ABSTRACT: Cultivation based assays combined with PCR or ELISA based methods to detect virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya and Bangladesh. The children were identified as either controls or cases with moderate to severe diarrhea using currently accepted clinical protocols. 3,610 stool samples were tested by established clinical culture techniques; 3,179 DNA samples by the Universal Biosensor® (Ibis Biosciences, Inc.); 1,466 DNA samples by GoldenGate® (Illumina, Inc.); and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected differing proportions of samples testing positive for each of seven enteric pathogens- enteroaggregative Escherichia coli (EAEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), Shigella, Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. Comparisons among detection methods included the frequency of positive stools and kappa values to make pair wise comparisons. Overall, standard culture methods detected Shigella, EPEC, ETEC and EAEC in a smaller proportion of the samples than either of the methods based on detection of the virulence genes from DNA in whole stool. The GoldenGate® method revealed the greatest agreement with the most other methods. Agreement among methods was higher in cases than controls. New molecular technologies have high potential for highly sensitive identification of bacterial diarrhea pathogens.
[Show abstract][Hide abstract] ABSTRACT: We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
[Show abstract][Hide abstract] ABSTRACT: In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification
of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly
automated. The strain typing approach is based on high-performance mass spectrometry, which “weighs” PCR amplicons with enough
mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's).
Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional
MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.
[Show abstract][Hide abstract] ABSTRACT: We describe a new approach to the sensitive and specific identification of bacteria, viruses, fungi, and protozoa based on broad-range PCR and high-performance mass spectrometry. The Ibis T5000 is based on technology developed for the Department of Defense known as T.I.G.E.R. (Triangulation Identification for the Genetic Evaluation of Risks) for pathogen surveillance. The technology uses mass spectrometry—derived base composition signatures obtained from PCR amplification of broadly conserved regions of the pathogen genomes to identify most organisms present in a sample. The process of sample analysis has been automated using a combination of commercially available and custom instrumentation. A software system known as T-Track manages the sample flow, signal analysis, and data interpretation and provides simplified result reports to the user. No specialized expertise is required to use the instrumentation. In addition to pathogen surveillance, the Ibis T5000 is being applied to reducing health care—associated infections (HAIs), emerging and pandemic disease surveillance, human forensics analysis, and pharmaceutical product and food safety, and will be used eventually in human infectious disease diagnosis. In this review, we describe the automated Ibis T5000 instrument and provide examples of how it is used in HAI control.
Journal of the Association for Laboratory Automation 12/2006; 11(6):341-351. DOI:10.1016/j.jala.2006.09.001 · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.
[Show abstract][Hide abstract] ABSTRACT: Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.
Proceedings of the National Academy of Sciences 06/2005; 102(22):8012-7. DOI:10.1073/pnas.0409920102 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work, we describe a strategy for the detection and characterization of microorganisms associated with a potential biological warfare attack or a natural outbreak of an emerging infectious disease. This approach, termed TIGER (Triangulation Identification for the Genetic Evaluation of Risks), relies on mass spectrometry-derived base composition signatures obtained from PCR amplification of broadly conserved regions of the microbial genome(s) in a sample. The sample can be derived from air filtration devices, clinical samples, or other sources. Core to this approach are “intelligent PCR primers” that target broadly conserved regions of microbial genomes that flank variable regions. This approach requires that high-performance mass measurements be made on PCR products in the 80–140 bp size range in a high-throughput, robust modality. As will be demonstrated, the concept is equally applicable to bacteria and viruses and could be further applied to fungi and protozoa. In addition to describing the fundamental strategy of this approach, several specific examples of TIGER are presented that illustrate the impact this approach could have on the way biological weapons attacks are detected and the way that the etiologies of infectious diseases are determined. The first example illustrates how any bacterial species might be identified, using Bacillus anthracis as the test agent. The second example demonstrates how DNA-genome viruses are identified using five members of Poxviridae family, whose members includes Variola virus, the agent responsible for smallpox. The third example demonstrates how RNA-genome viruses are identified using the Alphaviruses (VEE, WEE, and EEE) as representative examples. These examples illustrate how the TIGER technology can be applied to create a universal identification strategy for all pathogens, including those that infect humans, livestock, and plants.
International Journal of Mass Spectrometry 03/2005; 242(1-242):23-41. DOI:10.1016/j.ijms.2004.09.014 · 1.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.
[Show abstract][Hide abstract] ABSTRACT: Fourier transform ion cyclotron resonance (FTICR) mass spectrometry represents a unique platform with which to study nucleic acids and non-covalent complexes containing nucleic acids moieties. In particular, systems in which very high mass measurement accuracy is required, very complex mixtures are to be analyzed, or very limited amounts of sample are available may be uniquely suited to interrogation by FTICR mass spectrometry. Although the FTICR platform is now broadly deployed as an integral component of many high-end proteomics-based research efforts, momentum is still building for the application of the platform towards nucleic acid-based analyses. In this work, we review fundamental aspects of nucleic acid analysis by FTICR, focusing primarily on the analysis of DNA oligonucleotides but also describing applications related to the characterization of RNA constructs. The goal of this review article is to give the reader a sense of the breadth and scope of the status quo of FTICR analysis of nucleic acids and to summarize a few recently published reports in which researchers have exploited the performance attributes of FTICR to characterize nucleic acids in support of basic and applied research disciplines including genotyping, drug discovery, and forensic analyses.
Mass Spectrometry Reviews 03/2005; 24(2):265-85. DOI:10.1002/mas.20016 · 7.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.
[Show abstract][Hide abstract] ABSTRACT: In this work we present variations on in-hexapole infrared multiphoton dissociation (IRMPD) for the characterization of modified oligonucleotides using an ESI-FTICR spectrometer. We demonstrate that IRMPD in the external ion reservoir provides a comprehensive series of fragments allowing thorough characterization of a wide range of oligonucleotides containing alternative backbones and 2' substitutions. An alternative pulse sequence is presented that allows alternating MS and IRMPD MS/MS spectra to be acquired on a chromatographic timescale without loss in ionization duty cycle. Ions are excited to a larger cyclotron radius such that they "dodge" the IR laser beam that travels through the center of the trapped ion cell and impinges on the external ion reservoir creating IRMPD fragments that will be detected in the next scan. An alternative approach for directing IR radiation into the external ion reservoir using a hollow fiber waveguide as a photon conduit is presented. This approach offers a simple and robust alternative to the previously utilized on-axis scheme and may allow effective implementation with lower power lasers owing to the inherent increase in power density achieved by focusing the nascent laser beam into the hollow fiber waveguide.
Journal of the American Society for Mass Spectrometry 01/2004; 14(12):1413-23. DOI:10.1016/j.jasms.2003.08.007 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel scheme for performing infrared multiphoton dissociation (IRMPD) is presented in which a hollow fiber waveguide (HFWG) is used to transmit IR radiation into the ion storage region of a mass spectrometer. Efficient dissociation of oligonucleotide and protein ions is demonstrated on an ESI-FTICR instrument in which IRMPD is performed in the external ion reservoir and on a quadrupole ion trap. Using a simple optical scheme consisting of a single focusing lens and an x, y translator, the 10.6-microm IR laser beam, initially 3.5 mm in diameter, is focused into the vacuum-sealed HFWG. The small internal diameter and the high transfer efficiency of the waveguide allow IR radiation of high power density to be employed for IRMPD. In studies performed on a quadrupole ion trap, a 500-microm-i.d. waveguide was used as a medium to transmit IR radiation directly through a 700-microm orifice in the ring electrode. Efficient IRMPD of both a 12-mer oligonucleotide and the protein melittin were performed at laser powers of 0.5 and 3.2 W, respectively.
[Show abstract][Hide abstract] ABSTRACT: 7-Deaza purine modified oligonucleotides with a repeating motif have been sequenced by slow heating methods illustrating the ability to alter the preferred unimolecular decomposition pathway. The modified oligonucleotides limited the number of product ions per repeat unit resulting in an increased signal-to-noise ratio for the tandem mass spectrometry data. Loss of 7-deaza purine nucleobases or 7-deaza purine related product ions were not observed. The results illustrate the importance of the N7 position of purine nucleobases for low energy gas-phase decomposition. FRAGMENT results showed that for the 3− charge state of the 16-mer oligonucleotides, 5′-(AATG)4-3′ and 5′-(c7Ac7ATG)4-3′, that the 7-deaza deoxyadenosine modified sequence had a 38% greater relative energy of activation for unimolecular decomposition when compared to the unmodified sequence. For large DNA molecules that have purine-rich repeat units, the multiple sequence sites per repeat can result in a substantial loss of sequence information over large areas due to a reduced signal-to-noise ratio of the product ion spectrum. The incorporation of 7-deaza purines can limit the number of product ions during gas phase sequencing while ensuring sufficient sequence coverage to localize mutations or polymorphisms housed within a specific repeat unit.
International Journal of Mass Spectrometry 08/2002; 219(1):139-150. DOI:10.1016/S1387-3806(02)00544-4 · 1.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The utility of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FIICR) mass spectrometry as a new approach for genotyping short tandem repeats (STRs) is demonstrated. STRs are currently valued as a powerful source of genetic information with repeats that range in structure from simple to hypervariable. Two tetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUM-TH01, a simple STR with nonconsensus alleles, and vWA, a compound STR with nonconsensus alleles. For HUM-TH01, the genotype (i.e., repeat number of each allele) was determined for each of 30 individuals using mass measurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derived from heterozygous individuals were identified by mass as heteroduplexes that had formed between nonhomologous strands. Mass measurement of the double-stranded vWA amplicon was not sufficient for determining whether the individual was homozygous for allele subtype 18 or 18' since the amplicons differ by only 0.99 Da. Therefore, single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequently digesting the bottom strand using lambda-exonuclease. Accurate mass measurements were obtained for the single-stranded amplicons using internal calibration and the addition of a correction factor to adjust for the natural variation of isotopic abundances, confirming that the individual is homozygous for allele 18. Our results clearly demonstrate that ESI-FTICR mass spectrometry is a powerful approach to characterize both simple and compound STRs beyond the capabilities of electrophoretic technologies.
[Show abstract][Hide abstract] ABSTRACT: High-mass accuracy is demonstrated using internal calibration for product ions produced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) of a 15-mer oligonucleotide, 5'-(CTG)5-3'. Internal calibration for this tandem MS experiment was accomplished using a dual electrospray ionization (ESI) source coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) utilizing hexapole accumulation and gated trapping. The pulse sequence entails injection, trapping, and gas-phase isolation of the precursor ion of interest followed by the SORI-CID of this ion and, subsequently, injection and trapping of the internal mass calibrant (i.e., poly(ethylene glycol) with a 1000 Da average mass). The product ions and the poly(ethylene glycol) ions are then simultaneously excited by a broadband frequency chirp excitation waveform and detected. This technique corrects for space-charge effects on the measurement of an ion's cyclotron frequency experienced when externally calibrated data are used. While external calibration for FTICR-MS can result in mass errors of greater than 100 ppm, this internal standardization method demonstrated significantly more consistent accurate mass measurements with average mass errors ranging from -1.2 to -3.2 ppm for the 15-mer oligonucleotide used in this study. This method requires limited modifications to ESI-FTICR mass spectrometers and is applicable for both positive and negative modes of ionization as well as other sample types (e.g., pharmaceuticals, proteins, etc.).
[Show abstract][Hide abstract] ABSTRACT: Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.
Rapid Communications in Mass Spectrometry 03/2001; 15(5):348-50. DOI:10.1002/rcm.234 · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Single- and double-strand breaks bearing 3'-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3'-phosphoglycolate substrates for repair studies, 5'-(32)P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3'-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies.
[Show abstract][Hide abstract] ABSTRACT: An 82-base-pair polymerase chain reaction (PCR) product was amplified from the tetranucleotide short tandem repeat locus within the human tyrosine hydroxylase gene. PCR amplification was carried out using 100 ng of human nuclear DNA obtained from an individual who is homozygotic for the 9.3 allele resulting in a 50.5 kDa amplicon. To generate sufficient material for these investigations, several reactions were pooled and subsequently purified and quantified using UV-vis spectrophotometry. A serial dilution was carried out from a 2 microM stock solution providing solution concentrations down to 5 nM. Measurements were made using hexapole accumulation and gated trapping strategies in a 4.7 Telsa Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) which facilitated detection of the amplicon at the attomole level when electrosprayed from a 5 nM solution with a single acquisition! The signal-to-noise ratio was determined to be 8.3 for the spectrum derived from the 5 nM solution using the magnitude-mode mass spectral peak height for the most abundant charge-state. This remarkable sensitivity for large PCR amplicons will dramatically improve the ability of electrospray ionization mass spectrometry to address important genetic questions for low copy number genes or when the amount of initial template is limited; the latter issue is commonly encountered in DNA forensics. Furthermore, these data represents over 2 orders of magnitude decrease in detection limits over other existing ESI-MS reports concerning PCR products, including those conducted using FTICR-MS.
Fresenius Journal of Analytical Chemistry 03/2001; 369(3-4):246-51. DOI:10.1007/s002160000612
[Show abstract][Hide abstract] ABSTRACT: A dual electrospray ionization (ESI) source employed with hexapole accumulation and gated trapping provides a novel method of using an internal standard to achieve high mass accuracies in Fourier transform ion cyclotron resonance mass spectrometry. Two ESI emitters are sequentially positioned in front of the heated metal capillary inlet by a solenoid fitted to an XYZ micromanipulator; one emitter contains the analyte(s) of interest and the other an internal standard. A 5 V transistor-transistor logic pulse from the data station controls the solenoid by means of a solid-state relay so that matching of spectral peak intensities (i.e., analyte and internal standard intensities) can be accomplished by adjusting the hexapole accumulation time for each species. Polythymidine, d(pT)18, was used as the internal standard for all studies reported here. The absolute average error for an internally calibrated 15-mer oligonucleotide (theoretical monoisotopic mass = 4548.769 Da) was -1.1 ppm (external calibration: 41 ppm) with a standard deviation of +/-3.0 ppm (external calibration: +/-24 ppm) for a total of 25 spectra obtained at various hexapole accumulation time ratios. Linear least squares regression analysis was carried out and revealed a linear dependence of the magnitudes of the peak height ratios (analyte/internal standard) vs. hexapole accumulation time ratios (analyte/internal standard) which is described by the following equation: y = 0.45 x - 0.02. The fitted line had a %RSD of the slope of 28% with an R2 of 0.93. The applicability of this methodology was extended to a polymerase chain reaction product with a theoretical average molecular mass of 50,849.20 Da. With the internal standard, d(pT)18, an absolute average error of -8.9 ppm (external calibration: 44 ppm) based on five measurements was achieved with a standard deviation of 11 ppm (external calibration: +/-36 ppm), thus illustrating this method's use for characterizing large biomolecules such as those encountered in genomics and proteomics related research.
Journal of the American Society for Mass Spectrometry 11/2000; 11(10):876-83. DOI:10.1016/S1044-0305(00)00160-4 · 2.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.
The Analyst 05/2000; 125(4):619-26. DOI:10.1039/A908022H · 4.11 Impact Factor