Primary Polymerase Chain Reaction Research Articles

Molecular survey in relation to risk factors and haemato-biochemical alteration in Theileria equi infection of equines in Punjab Province, India.

Equine piroplasmosis caused by Theileria equi, an apicomplexan intracellular haemoprotozoan parasite effects equids throughout the world. Current investigation is the first detailed epidemiological survey report based on conventional (blood smear) and primary polymerase chain reaction (PCR) for the detection of T. equi on 464 equids (426 horses and 38 donkeys/mules) in Punjab province of India. PCR targeting 18S rRNA gene of T. equi produced high fidelity 709bp amplification products with 100% concordance with blood films. The prevalence of infection was proportional (P≤0.01) to temperature and aridness of the regions, which turned out to be the most important physical factor pertaining to T. equi infection. Spatial distribution analysis revealed an increasing trend of disease prevalence from north-eastern to south-western region of Punjab by both the techniques. Haemato-biochemical analysis revealed significant decrease in total erythrocyte count and haemoglobin; and increase in mean corpuscular haemoglobin, serum globulin, glucose, creatinine, aspartate aminotransferase and bilirubin levels (P≤0.05). This study divulges the endemicity of equine theileriosis in Punjab with the prominence of various odds of infection, emphasising the pathophysiological manifestation associated with latent infection of T. equi detectable by PCR.

A vertically stacked, polymer, microfluidic point mutation analyzer: Rapid high accuracy detection of low-abundance K- ras mutations

Recognition of point mutations in the K- ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1 h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-μl sample, equivalent to DNA from 42 mutant cells.

High-Throughput Selection, Enumeration, Electrokinetic Manipulation, and Molecular Profiling of Low-Abundance Circulating Tumor Cells Using a Microfluidic System

A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s(-1), which allowed for the ability to process 1 mL of whole blood in <40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ± 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 μL min(-1) to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm(-1), the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 μL, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.

Microfluidic Reactor for Sequential Operation of Polymerase Chain Reaction/Ligase Detection Reaction

We have developed a microfluidic bioreactor for detecting single base mutations in genomic DNA, where a primary polymerase chain reaction (PCR) and an allelespecific ligation detection reaction (LDR) were conducted in sequence. The effect of carryover from the primary PCR on the subsequent LDR was thoroughly investigated in terms of LDR yield and fidelity, and we found that a post-PCR treatment for the amplicons prior to its incorporation into the LDR phase was not necessarily required, which allowed us to use a simple diffusive mixer to online mix the post- PCR solution with the LDR reagents. We also performed a numerical analysis to roughly estimate a channel length required for the diffusive mixing. We successfully demonstrated the ability of the system to detect one mutant DNA in 1000 normal sequences at a relatively high processing speed (total processing time = ca. 28.1 min).

Construction of internal control for the quantitative assay of Aspergillus fumigatus using real-time nucleic acid sequence-based amplification

We have developed a scheme for quantitative real-time (RTi) nucleic acid sequence-based amplification (NASBA) for the detection of Aspergillus fumigatus using internal control (IC) RNA. Construction of IC RNA began with the synthesis of nontarget sequences from Clavibacter michiganensis subsp. sepedonicus by a primary polymerase chain reaction (PCR) step, followed by a secondary PCR step using chimeric primers to produce a chimeric sequence including a T7 promoter region. Finally, chimeric IC RNAs were constructed by the use of in vitro transcription. The assay detected A. fumigatus within a range of 10 4 to 10 8 copies/mL of RNA and 10 0 to 10 8 cells. When the assay was performed with the target and IC RNA in 1 reaction in a single tube, there was little interference of the IC RNA in the measurement of the amount of target. The amount of RNA calculated using the assay was not significantly different from the amount of input RNA as indicated by Bland–Altman analysis. The IC RNA we constructed can be used in RTi-NASBA for quantitative detection of Aspergillus with good precision and accuracy.

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Cloning and expression of murine SC1, a gene product homologous to SPARC.

A number of cDNAs (SC1, QR1, and hevin) have been shown to be similar to SPARC (secreted protein acidic and rich in cysteine), a matricellular protein that regulates cell adhesion, cell cycle, and matrix assembly and remodeling. These proteins are 61-65% identical in the final 200 residues of their C-termini; their N-terminal sequences are related but more divergent. All have an overall acidic pl, with a follistatin-like region that is rich in cysteine, and a Ca+2 binding consensus sequence at the C-terminus. Using degenerate primers representing the most highly conserved region in SPARC, SC1, and QR1, we identified a 300-BP SC1 clone in a primary polymerase chain reaction (PCR) screen of a mouse brain cDNA library. This cDNA was used to obtain a full-length clone, which hybridized to a 2.8-KB RNA abundant in brain. Mouse SC1 displays a similarity of 70% to mouse SPARC at the amino acid level. Northern blot and RNAse protection assays revealed a 2.8-KB mRNA expressed at moderate levels (relative to brain) in mouse heart, adrenal gland, epididymis, and lung, and at low levels in kidney, eye, liver, spleen, submandibular gland, and testis. In contrast to SPARC, in situ hybridization showed expression of SC1 mRNA in the tunica media and/or adventitia of medium and large vessels; transcripts were not detected in capillaries, venules, or large lymphatics. The distribution of transcripts for SC1 was also different from that of SPARC in several organs, including adrenal gland, lung, heart, liver, and spleen. Moreover, SC1 mRNA was not evident in endothelium cultured from rat heart, bovine fetal and adult aorta, mouse aorta, human omentum, and bovine retina. Cultured smooth muscle cells and fibroblasts also failed to express SC1 mRNA. The absence of SC1 transcript in cultured cells indicates that the SC1 gene is potentially sensitive to regulatory factors in serum or to a three-dimensional architecture conferred by the extracellular matrix that is lacking in vitro. In conclusion, the expression of SPARC and SC1 appears to be coincident in specific tissues (e.g., adrenal gland and brain), but these proteins exhibit distinct expression patterns in most organs of the mouse. Because SC1 and SPARC are structurally similar and exhibit counteradhesive effects on cultured cells, their overlapping and/or adjacent expression in most tissues predicts that one protein might compensate functionally, at least in part, for the other.

Profile of immunoglobulin heavy chain variable gene repertoires and highly selective detection of malignant clonotypes in acute lymphoblastic leukemia

The predominant B cell immunoglobulin heavy chain variable gene (IgH-V) usage and the uniquely rearranged, clonotype-specific variable-diversity-joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B-ALL) using a novel DNA-based gene amplification strategy. The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones. An initial primary polymerase chain reaction (PCR), directed by an IgH-J generic primer and a complement of family-specific IgH-V primers, defined the major B cell IgH-V gene usage. Use of an IgH-J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription-PCR amplification method. Monoclonality of rearranged VDJ bands was further substantiated by high-resolution denaturant gel electrophoretic analysis. The predominant amplified bands were subcloned and sequenced. By sequencing through VDJ juxtaposed regions, that is, the third complementarity-determining region, clonotype-specific primers were developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone. Analysis of the products of the CPD-PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage. Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B-ALL studied. Two or more clonal IgH-VDJ amplified sequences were observed in 50% of the B-ALL bone marrows analyzed. In two cases, clonotype-specific oligodeoxynucleotide primers, derived from B-ALL VDJ sequences, directed the secondary CPD-PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation.