Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry

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 μM 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.     

Conductivity detection of polymerase chain reaction products separated by micro-reversed-phase liquid chromatography

In this paper, we describe the application of micro-reversed-phase high-performance liquid chromatography (mu-RP-HPLC) for the separation and/or purification of polymerase chain reaction (PCR) products with detection accomplished using a miniaturized conductivity detector. The conductivity detector used two Pt wires and a bipolar waveform applied to the electrode pair from which the conductivity of the bulk solution could be measured. In the mobile phase used for the mu-RP-HPLC separation of the PCR product, the mass detection limit for herring sperm DNA using conductivity was found to be 11 ng. Efficient separation of the PCR amplicon from the other reagents present in the PCR cocktail was achieved in less than 4 min with a capacity factor of 2.5 and separation efficiency of 9.1 x 10(3) plates. The separation was carried out using reversed-phase ion-pair chromatography with a triethylammonium acetate ion-pairing agent.     

Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 10⁵ times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 10³ CFU mL⁻¹  S. pneumoniae.     

High-sensitive determination of human alpha-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis

ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5 nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400 nM and the LOD was 2 nM (74 ng/mL, S/N = 3), i.e., 40 amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin.     

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

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.     

Electrochemical probe DNA design in PCR amplicon sequence for the optimum detection of microbiological diseases

Direct electrochemical genosensor was developed for the detection of a probe sequence relative position in a PCR amplicon for the optimum detection of bacterial and microbiological diseases, in this study. The genosensor relies on a label-free electrochemical detection. The amino-linked inosine modified (guanine-free) coequal capture probes which were chosen from different parts of a PCR amplicon, immobilized on to disposable pencil graphite electrodes (PGE) by electrostatically and covalently. As a model case Hepatitis B virus (HBV) genome amplicon was used for the detection and specification. Hybridization was occurred after surface coverage with denatured amplicons. After hybridization, optimum probe sequence position was identified by using the differences between the responses of guanine oxidation signals. The results of this study might have a great convenience for the microbiological diseases detection applications such as DNA micro arrays.     

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.     

Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

A new genosensor for meningococcal meningitis diagnosis using biological samples

In this work, a new electrochemical biosensor for DNA detection of bacterial meningitis is proposed. The system is based on specific DNA fragments from the Neisseria meningitidis genome as a probe incorporated on graphite electrodes modified with poly(4-aminophenol). Detection of a complementary oligonucleotide sequence, a specific 710-base pair amplicon, and the genomic DNA of bacteria was carried out by differential pulse voltammetry, using ethidium bromide as an electroactive indicator of hybridization. The complementary oligonucleotide and the genomic DNA of Neisseria meningitidis were quantified by the genosensor, showing detection limits of 0.6 ng μL^?1 and about 6 ng μL^?1, respectively. Morphological differences were observed between hybridized and unhybridized surfaces by atomic force microscopy. The biosensor showed high selectivity, discriminating non-specific targets, and high stability retaining over 98% of its original activity after 120 days of storage. The bioelectrode was effective in discriminating the genomic DNA in samples with human serum without significant interference, proving to be an interesting platform for meningococcal meningitis diagnosis.     

Label-free selective DNA detection with high mismatch recognition by PNA beacons and ion exchange HPLC

Two 11mer peptide nucleic acid (PNA) beacons were synthesized and tested for the detection of full-matched or single mismatched DNA. Fluorescent measurements carried out in solution showed only partial discrimination of the mismatched sequence, while using anion-exchange HPLC, in combination with fluorimetric detection, allowed DNA analysis to be performed with high sensitivity and extremely high sequence selectivity. Up to >90 : 1 signal discrimination in the presence of one single mismatched base was observed. The analysis was tested on both short and long DNA oligomers. Detection of DNA obtained from PCR amplification was also performed allowing the selective detection of the target sequence in complex mixtures. Label free detection of the DNA with high sequence selectivity is therefore possible using the present approach.     

An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10^–12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

CEPH family 1362 STR database: An online resource for characterization of PCR products using electrospray ionization mass spectrometry

An online database has been established in order to validate electrospray ionization mass spectrometry (ESI-MS) for genotyping and to publicize the procedures developed in our laboratory for the characterization of PCR products by ESI-MS. Genotypes derived from short tandem repeat (STR) loci that were obtained using ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) have been posted for fifteen members of the CEPH family 1362 pedigree. The website provides specific information such as PCR parameters, PCR product cleanup approaches, and ESI solution compositions to enable other laboratories to reproduce our data. Links are provided to related websites in an effort to integrate information regarding the CEPH family, STR genotyping, and mass spectrometry. The database, currently available at http://www.people.vcu.edu/ -dcmuddim/genotype/ will be routinely updated with genotypes from additional STR loci including PCR parameters as well as PCR cleanup strategies as further developments are completed.     

Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar–attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms.

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.     

Aptamer-functionalized solid phase microextraction–liquid chromatography/tandem mass spectrometry for selective enrichment and determination of thrombin

In this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human α-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC–MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC–MS/MS provided a good linear dynamic range of 0.5–50 nM in diluted human plasma with a good correlation coefficient (R² = 0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC–MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.     

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.     

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease

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.     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.