Computational thermal analysis of a continuous flow micro Polymerase Chain Reaction (PCR) chip

The first continuous flow micro PCR introduced in 1998 has attracted considerable attention for the past several years because of its ability to amplify DNA at much faster rate than the conventional PCR and micro chamber PCR method. The amplification is obtained by moving the sample through 3 different fixed temperature zones. In this paper, the thermal behavior of a continuous flow PCR chip is studied using commercially available finite element software. We study the temperature uniformity and temperature gradient on the chip’s top surface, the cover plate and the interface of the two layers. The material for the chip body and cover plate is glass. The duration for the PCR chip to achieve equilibrium temperature is also studied.

Optimisation and analysis of three polymerase chain reaction (PCR) systems for the detection of environmentally significant microorganisms

Conventional assessments of water quality are based on the determination of bacterial indicator numbers. The detection of an indicator organism only provides presumptive evidence of the presence of harmful organisms By using polymerase chain reaction (PCR) to monitor water quality, indicators and pathogenic organisms can be specifically detected with a high sensitivity. The research illustrates the importance of optimisation and the maintenance of reaction efficiency in the accuracy and precision of the PCR.

A disposable multiplex polymerase chain reaction (PCR) chip and device

Malaysian patent application number PCT/MY2008/000190 Australian application number : 2009203047

Development and application of polymerase chain reaction-based assays for Rathayibacter Toxicus and a Bacteriophage associated with annual ryegrass (Lolium Rigidum) toxicity

Annual ryegrass toxicity (ARGT) is responsible for significant stock losses in South Australia and Western Australia. The toxicity is caused by corynetoxins produced by the bacterium Rathayibacter toxicus (with the possible involvement of a bacteriophage), which infects annual ryegrass (Lolium rigidum). Polymerase chain reaction (PCR)-based assays, compatible with an existing enzyme-linked immunosorbent assay for the corynetoxins, have been developed and used to screen L. rigidum for both the presence of R. toxicus and for the bacteriophage isolate NCPPB 3778. The results from analysing bacterially infected galls from toxic grain screenings showed a positive correlation between the presence of the bacterium and corynetoxins but not with the bacteriophage. Analysis of pasture-derived samples of annual ryegrass showed about a 50% correlation of corynetoxins with bacterial presence and about a 5% correlation of phage with the presence of the bacterium. These observations support the potential application of the PCR-based assays in providing a useful, complementary tool in the assessment of the likelihood of pasture and feed to cause ARGT and to enable a better understanding of the complex aetiology of ARGT.

Design, fabrication and evaluation of universal polymerase chain reaction devices

Novel and low cost PCR devices were developed to perform rapid polymerase chain reaction based diagnostics for infectious diseases. The main advantages of using these devices are that it can perform multi sample and also multiplexing of DNA samples woth low sample volume compared to the conventional PCR devices.

Rapid multi sample DNA amplification using rotary-linear polymerase chain reaction device (PCRDisc)

Multiple sample DNA amplification was done by using a novel rotary-linear motion polymerase chain reaction (PCR) device. A simple compact disc was used to create the stationary sample chambers which are individually temperature controlled. The PCR was performed by shuttling the samples to different temperature zones by using a combined rotary-linear movement of the disc. The device was successfully used to amplify up to 12 samples in less than 30 min with a sample volume of 5 μl. A simple spring loaded heater mechanism was introduced to enable good thermal contact between the samples and the heaters. Each of the heater temperatures are controlled by using a simple proportional–integral–derivative pulse width modulation control system. The results show a good improvement in the amplification rate and duration of the samples. The reagent volume used was reduced to nearly 25% of that used in conventional method.

Expression of Na+/H+ exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis

Sodium/proton exchangers (NHE) are transmembrane proteins that facilitate the exchange of a Na⁺ ion for a H⁺ ion across cellular membranes. The NHE are present in the gills of fishes and are believed to function in acid-base regulation by driving the extrusion of protons across the branchial epithelium in exchange for Na⁺ in the water. In this study, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the presence of a branchial NHE in the gills of the Atlantic hagfish, Myxine glutinosa. The subsequent partial cDNA sequence shares homology with other vertebrate and invertebrate NHE isoforms. In addition, using semi-quantitative, multiplex RT-PCR we demonstrate that mRNA expression of hagfish gill NHE is upregulated following an induced metabolic acidosis. Expression was increased to 4.4 times basal levels at 2-h post-infusion and had decreased to 1.6 times basal by 6 h. Expression had returned to basal levels by 24-h post-infusion. The inference from this study is that a gill NHE which is potentially important in acid-base regulation has been present in the vertebrate lineage since before the divergence of the hagfishes from the main vertebrate line.

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish, Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.

The taq IA and Ser311Cys polymorphisms in the dopamine D2 receptor gene and obesity

Dopamine D2 receptors (DRD2) in the central nervous system are involved in the regulation of feeding. It remains to be elucidated if mutations in the DRD2 gene contribute to the development of obesity. The aim of the present study was to investigate whether the Taq IA and Ser311Cys polymorphisms in the DRD2 gene are associated with obesity in Nauruan and Australian subjects. Subjects were selected based on extremes of the body mass index (BMI) distribution. Two groups of Australian women were selected. The leanest group had a mean BMI of 22.5 kg/m2 (range: 20.3-24.3) and the heaviest group had a mean of 36.1 kg/m2 (32.5-44.1). Four groups of Nauruan subjects were selected. Leanest men had a mean BMI of 33.0 kg/m2 (28.4-36.9), heaviest men had a mean of 52.8 kg/m2 (46.5-69.2), leanest women had a mean of 34.8 kg/m2 (28.2-41.8) and heaviest women had a mean of 55.1 kg/m2 (49.3-73.8). Subjects were genotyped for the Taq IA and Ser311Cys polymorphisms using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and allelic discrimination TaqmanTM PCR respectively. Leanest and heaviest groups were examined for differences in genotype frequency. Taq IA and Ser311Cys genotype frequencies did not differ significantly between leanest and heaviest Nauruan groups, or between leanest and heaviest Australians. Haplotype frequencies of these polymorphisms did not differ between leanest and heaviest groups. The Taq IA and Ser311Cys polymorphisms in the DRD2 gene are unlikely to be common causes of obesity in these populations.

Relationship of glutathione S-transferase genotypes with side-effects of pulsed cyclophsphamide therapy in patients with systemic lupus erythematosus

Aims
Cyclophosphamide (CTX) is an established treatment of severe systemic lupus erythematosus (SLE). Cytotoxic CTX metabolites are mainly detoxified by multiple glutathione S-transferases (GSTs). However, data are lacking on the relationship between the short-term side-effects of CTX therapy and GST genotypes. In the present study, the effects of common GSTM1, GSTT1, and GSTP1 genetic mutations on the severity of myelosuppression, gastrointestinal (GI) toxicity, and infection incidences induced by pulsed CTX therapy were evaluated in patients SLE.
Methods
DNA was extracted from peripheral leucocytes in patients with confirmed SLE diagnosis (n = 102). GSTM1 and GSTT1 null mutations were analyzed by a polymerase chain reaction (PCR)-multiplex procedure, whereas the GSTP1 codon 105 polymorphism (Ile→Val) was analyzed by a PCR-restriction fragment length polymorphism (RFLP) assay.
Results
Our study demonstrated that SLE patients carrying the genotypes with GSTP1 codon 105 mutation [GSTP1*-105I/V (heterozygote) and GSTP1*-105 V/V (homozygote)] had an increased risk of myelotoxicity when treated with pulsed high-dose CTX therapy (Odds ratio (OR) 5.00, 95% confidence interval (CI) 1.96, 12.76); especially in patients younger than 30 years (OR 7.50, 95% CI 2.14, 26.24), or in patients treated with a total CTX dose greater than 1.0 g (OR 12.88, 95% CI 3.16, 52.57). Similarly, patients with these genotypes (GSTP1*I/V and GSTP1*V/V) also had an increased risk of GI toxicity when treated with an initial pulsed high-dose CTX regimen (OR 3.33, 95% CI 1.03, 10.79). However, GSTM1 and GSTT1 null mutations did not significantly alter the risks of these short-term side-effects of pulsed high-dose CTX therapy in SLE patients.
Conclusions
The GSTP1 codon 105 polymorphism, but not GSTM1 or GSTT1 null mutations, significantly increased the risks of short-term side-effects of pulsed high-dose CTX therapy in SLE patients. Because of the lack of selective substrates for a GST enzyme phenotyping study, timely detection of this mutation on codon 105 may assist in optimizing pulsed high-dose CTX therapy in SLE patients.

Genetic polymorphisms of cytochrome P450 2B6 gene in Han Chinese

Cytochrome P450 (CYP2B6) is an important enzyme that metabolizes more than eight compounds and about 3.0% of therapeutic drugs. The genetic polymorphisms of CYP2B6 have earlier been studied in Caucasian, Japanese and Korean, but the data are lacking for Han Chinese. The aim of this study was to investigate the frequencies of allelic variants of CYP2B6 in healthy Han Chinese and compare with those in other ethnic groups reported in the literature. Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) method was used to test the five common non-synonymous single nucleotide polymorphisms (SNPs) of CYP2B6 gene, namely, 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T in unrelated healthy Han Chinese (n = 193). The study demonstrated that the frequencies of 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T SNPs in Han Chinese were 0.03, 0.21, 0, 0.28 and 0.003, respectively. The frequencies of all five SNPs tested in female were higher than those in male, but the statistical difference was insignificant (P > 0.05). Compared to the data reported in the literature, the frequencies of common CYP2B6 allelic variants in Chinese are similar to those of other Asian populations including Japanese and Korean, but markedly different from those in Caucasians. These results indicate the presence of marked ethnic difference in CYP2B6 SNP frequencies between Chinese and Caucasian. Further studies are required to explore the impact of these SNPs of CYP2B6 gene on the clinical response (efficacy and toxicity) to drugs that are substrates for CYP2B6 in patients.

Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF

Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel–nitrilotriacetic acid (Ni–NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.