Multiplex amplified product-length polymorphism analysis of 36 mitochondrial single-nucleotide polymorphisms for haplogrouping of East Asian populations

We present a reliable, rapid, and economical multiplex amplified product-length polymorphism (APLP) method for analyzing the haplogroup-diagnostic mitochondrial single-nucleotide polymorphisms (mtSNPs) in East Asian populations. By examining only 36 haplogroup-specific mtSNPs in the coding region by using four 9-multiplex polymerase chain reaction (PCR) and subsequent electrophoresis, we could safely assign 1815 individuals from 8 populations of Japanese, Korean, Chinese, and Germans to 45 relevant haplogroups. This multiplex APLP analysis of coding-region mtSNPs for haplogrouping is especially useful not only for molecular phylogenetic studies but also for large-scale association studies due to its rapid and economical nature. This is the first panel of mtSNPs in the coding region to be used for haplogrouping of East Asian populations.     

Multiplex primer extension analysis for rapid detection of major European mitochondrial haplogroups

The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n = 1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility.     

A cationic tetrahedral chromophore for amplified DNA detection

The tetrahedral cationic chromophore, tetrakis [4-(9,9-bis(6′-(N,N,N-trimethylammonium)hexyl)-2-fluorenyl)phenyl]methane (1) shows better fluorescence resonance energy transfer (FRET) to the fluorescein (Fl) attached to the 5′-terminus of double-stranded DNA (dsDNA-Fl) as compared to the linear oligomers 2 and 3 and also provides efficient DNA hybridization detection.     

Multiplex pyrosequencing of InDel markers for forensic DNA analysis

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator^® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator^® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof‐of‐principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost‐effective alternative for some applications.     

Redox-active nucleic-acid replica for the amplified bioelectrocatalytic detection of viral DNA

A new concept for the amplified electrochemical detection of the 7229-base viral DNA of M13phi is developed. A thiolated 27-base nucleic acid (1) is assembled on an Au-electrode. Hybridization between the sensing interface and the M13phi DNA is followed by the polymerase-induced replication of the analyte DNA in the presence of dCTP, dGTP, dATP, and ferrocene-tethered-dUTP (2). The generated redox-active replica mediates electron transfer between the enzyme glucose oxidase (GOx) and the electrode and activates the bioelectrocatalyzed oxidation of glucose. The bioelectrocatalyzed oxidation of glucose provides a biocatalytic amplification path for the formation of the redox-active replica. The electrochemical techniques to follow the replication and the bioelectrocatalytic amplification are differential pulse voltammetry and cyclic voltammetry. The electrical responses from the system relate to the bulk concentration of the M13phi DNA, thus enabling the quantitative analysis of the viral gene.     

Microfluidic chip-based aptasensor for amplified electrochemical detection of human thrombin

In this paper, a microchip-based sandwich-type aptasensor is developed for the detection of human thrombin. The SH-aptamer/thrombin/alkaline phosphatase-functionalized aptamer (ALP-aptamer) system was constructed in the microfluidic channels. And the substrate solution containing 4-aminophenyl phosphate (p-APP) was introduced to the microchannels for the end-column electrochemical detection. The on-chip aptasensor has a broad linear response range of 1–100 pM with a detection limit of 1 pM, which shows high sensitivity and specificity. The system was then applied to detect thrombin in human serum sample. Therefore, the on-chip aptasensor has a great promise for detecting and screening ultratrace levels of biomarkers in the complex matrices.     

Genotyping Taenia tapeworms by single-strand conformation polymorphism of mitochondrial DNA.

To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a single-strand conformation polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP-based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.     

Carbon-nanotube-modified glassy carbon electrodes for amplified label-free electrochemical detection of DNA hybridization

The preparation and attractive performance of carbon-nanotube modified glassy-carbon (CNT/GC) electrodes for improved detection of purines, nucleic acids, and DNA hybridization are described. The surface-confined multiwall carbon-nanotube (MWCNT) facilitates the adsorptive accumulation of the guanine nucleobase and greatly enhances its oxidation signal. The advantages of CNT/GC electrodes are illustrated from comparison to the common unmodified glassy carbon, carbon paste and graphite pencil electrodes. The dramatic amplification of the guanine signal has been combined with a label-free electrical detection of DNA hybridization. Factors influencing the enhancement of the guanine signal are assessed and optimized. The performance characteristics of the amplified label-free electrochemical detection of DNA hybridization are reported in connection to measurements of nucleic-acid segments related to the breast-cancer BRCA1 gene.     

Terahertz-time domain spectroscopy for the detection of PCR amplified DNA in aqueous solution

In this work we present a label free quantitative detection method for DNA samples amplified by polymerase chain reaction (PCR) in aqueous medium using terahertz-time domain spectroscopy (THz-TDS) in the frequency range from 0.3 to 1.2 THz. The DNA samples of 133 and 697 base pairs were prepared using PCR. We measured the absorption coefficients of DNA solutions in the concentration range of 0-0.3 ng μl(-1). For both DNA types, the absorption coefficients decreased with increasing DNA concentrations. The average change in absorption coefficients compared to buffer within the frequency range of 0.8-1.0 THz showed a linear behavior. Our results demonstrate that THz-TDS can detect PCR amplified DNA in aqueous solution with a minimum concentration of 0.1 ng μl(-1) and a minimum sample volume of 10 μl.     

A microfluidic system for fast detection of mitochondrial DNA deletion

This study reports an integrated microfluidic system capable of automatic extraction and analysis of mitochondrial DNA (mtDNA). Mitochondria are the energy production and metabolism centres of human and animal cells, which supply most of the energy for maintaining physiological functions and play an important role in the process of cell death. Because it lacks an effective repair system, mtDNA suffers much higher oxidative damage and usually harbours more mutations than nuclear DNA. Alterations of mtDNA have been reported to be strongly associated with mitochondrial dysfunction, mitochondria-related diseases, aging, and many important human diseases such as diabetes and cancers. Thus, an effective tool for automatic detection of mtDNA deletion is in great need. This study, therefore, proposed a microfluidic system integrating three enabling modules to perform the entire protocol for the detection of mtDNA deletion. Crucial processes which included mtDNA extraction, nucleic acid amplification, separation and detection of the target genes were automatically performed. When compared with traditional assays, the developed microfluidic system consumed fewer samples and reagents, achieved a higher mtDNA extraction rate, and could automate all the processes within a shorter period of time (150 minutes). It may provide a powerful tool for the analysis of mitochondria mutations in the near future.     

Electrophoretic detection of genetic variability among Schistosoma japonicum isolates by sequence-related amplified polymorphism

In the present study, sequence‐related amplification polymorphism (SRAP) was utilized to study the genetic variability among Schistosoma japonicum isolates from different provinces in China, using Schistosoma mansoni from Puerto Rico for comparison. Five out of ten tested SRAP primer combinations displayed significant polymorphisms among S. japonicum isolates from China, namely ME2/EM1, ME4/EM1, ME4/EM6, ME5/EM4 and ME5/EM5. Analysis of the 61 S. japonicum samples from China with five SRAP primer combinations identified a total of 83 reproducible polymorphic fragments. The number of fragments using each primer combination ranged from 14 to 19, with an average of 16 polymorphic bands per primer pair, and the size of fragment ranged approximately from 100 to 1000 bp. Representative‐specific SRAP fragments were excised from the gels, and confirmed by PCR amplification of genomic DNA using primers designed and based on the sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram was constructed. UPGMA clustering algorithm categorized S. japonicum isolates from China into nine clades and two lineages (representing the mountainous and lake/marshland regions). These results indicate the usefulness of the SRAP technique for revealing genetic variability among S. japonicum isolates from China, and the SRAP technique should be applicable to other living organisms.     

An amplified single-walled carbon nanotube-mediated chemiluminescence turn-on sensing platform for ultrasensitive DNA detection

We have developed an amplified chemiluminescence turn-on sensing platform that relies on single-walled carbon nanotubes for ultrasensitive DNA detection. This new type of assay exhibits higher detection sensitivity over traditional biosensors by three orders of magnitude and high specificity for the target molecules.     

Enzyme-free amplified detection of DNA by an autonomous ligation DNAzyme machinery

The Zn(2+)-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10(-11) M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Capillary electrophoresis with dual laser detection in separation of amplified fragment length polymorphism fragments

We are presenting the application of CE technique with dual‐channel LIF detection for the simultaneous separation of DNA fragments labeled with two different fluorescence dyes. The optimal conditions of the analysis were determined for the separation of amplified fragment length polymorphism (AFLP) fragments labeled with 5′‐6‐carboxyfluorescein (6‐FAM) and the DNA size standard labeled with sulfoindocyanine succinimidyl ester (Cy‐5). CE equipped with both argon ion and diode lasers is a good alternative for sequencers and might be applied in analyses of PCR products generated by various fingerprinting methods.     

A universal platform for amplified multiplexed DNA detection based on exonuclease III-coded magnetic microparticle probes

An amplified multiplexed DNA detection biosensor has been developed, which combines the unique cleavage function of exonuclease III (Exo III) with the separating ability of magnetic microparticles (MMPs). By using different fluorophores, the multiplexed detection of DNA is demonstrated.     

Scanning positional variations in single-nucleotide polymorphism of DNA: an electrochemical study

While there are a number of electrochemical methods reported that enable the detection of single nucleotide mismatches, the determination of mismatch position in a double stranded DNA remains an unsolved challenge. Using a model system, we systematically explored the electrochemical response of all possible positions of single nucleotide mismatches in a set of 25-mer DNA films. These ds-DNA sequences each with a single mismatch at one of the twenty-five positions were bound to gold surfaces through a Au-S linkage and analyzed by electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) in the absence and presence of Zn(2+). We expected a unique response from each mismatched sequence in order to discriminate the mismatch positions. A pattern emerges between the electrochemical signals and mismatch positions. The positions can be grouped broadly into positions that exhibit large differences between matched and mismatched DNA (around positions 5 and 9) and those that exhibit smaller differences (around positions 1, 13 and 23) in the charge transfer resistance ΔR(ct), evaluated by EIS, and the apparent rate constant k(0), evaluated by SECM. To the best of our knowledge, this is the first study evaluating the electrochemical response of a single nucleotide mismatch as a function of mismatch positions along an oligonucleotide sequence.