The Effect of Input DNA Copy Number on Genotype Call and Characterising SNP Markers in the Humpback Whale Genome Using a Nanofluidic Array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue

Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification

ABSTRACT: Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16 % through to 99.6 % saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5 % was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies

Characterization of arsenic resistant bacteria and a novel gene cluster in \u27Bacillus\u27 sp. CDB3

Arsenic is a toxic metalloid of the nitrogen family which is found in both natural environments and sites contaminated in a number of ways. Arsenate acts as a phosphate analog which interferes with phosphate uptake and utilization and arsenite disrupts enzymatic function. Micro organisms have evolved a variety of mechanisms in coping with toxicity of arsenic and the best known detoxification pathway involves the arsenic resistance (ars) cluster. To date, a number of ars clusters have been characterized at the molecular level. Contamination of cattle dip-site soils with arsenic along with other toxicants such as DDT represents a major pollution problem in agricultural sites and has been shown to have distinct effects on the soil microbial populations. Little is known of the micro organisms at these sites. This thesis investigated the identity of some of the rhizosphere bacteria isolated from arsenic contaminated dip-site soils of north-eastern New South Wales and functionally characterized some of the ars genes cloned from one strain. Five bacterial strains isolated from cattle dip-sites (referred to as CDB) were identified based on morhological, biochemical and 16S rDNA sequence characters. These bacterial strains belonged to four different genera, CDB1- Arthrobacter sp., CDB2- Ochrobactrum sp., CDB3 & CDB4- Bacillus spp and CDB5- Serratia sp. The arsenic resistance profiles of these bacteria were quite different. The highest resistance to arsenite was by CDB5 followed by CDB4, CDB3, CDB2 and CDB1. CDB2 was exceptionally tolerant to arsenate, exhibiting normal growth on agar containing 200 mM arsenate, an almost saturated concentration, while all the other strains showed a minimum inhibitory concentration to arsenate of 75 mM. From Bacillus sp. CDB3, a novel ars gene cluster was cloned and sequenced (Luo, 2006), which revealed eight intact open reading frames organised in a unique gene order, arsRYCDATIP when compared to other ars gene clusters. This gene cluster was found to exist on the chromosome, unlike the arsRDABC clusters which exist on plasmids of Gram negative bacteria. Bioinformatic analysis was undertaken to examine any novel sequence characters. Alignment of the CDB3 ArsD with known ArsDs showed that the CDB3 ArsD protein lacks the four C-terminal cysteine residues (Cys112-Cys113 and Cys119- Cys120), which have been demonstrated to be required for induction in E. coli pR773 ArsD, suggesting the regulatory mechanism by CDB3 ArsD may be different from that of E. coli pR773. This was the case, when the ArsD of CDB3 was mutated and compared to the intact ArsD as presented in this thesis, the E. coli cells bearing mutant ArsD showed sensitivity to arsenic. The membrane bound AsIII pump encoded by the second ORF is a YqcL type protein but may still associate with an ATPase encoded by the fifth ORF. Phylogenetic analysis of arsenite efflux pumps also revealed the existence of a sub-group of the ArsB group proteins, adding to the diversity of the arsenite pump protein family. Further investigation was done to determine whether the CDB3 ArsA, (which is known to couple to ArsB) can couple to YqcL and vice versa. Results indicated that CDB 3 ArsA can couple to both YqcL and E. coli ArsB resulting in elevated resistance to the host, confirming the functionality of ArsA. No data on ArsA coupling with YqcL is available and this thesis was the first instance to show the interaction of YqcL with ArsA in extruding arsenite out of the cell. Transcriptional regulation of CDB3 ars gene cluster was performed by reporter gene and northern analysis. Northern analysis showed that the mRNA transcription could read through the whole cluster, indicating that the CDB3 ars gene cluster can be transcribed under the control of a single promoter. However, some short transcripts were also detected. These results showed that the ars gene cluster can be transcribed as whole cluster, but under certain conditions it may be transcribed as two or more sub-operons. This was further studied by the reporter gene analysis which indicated active promoters in front of arsR, arsD and arsT. Gel mobility shift assays also displayed the existence of a promoter in front of arsD, where the purified ArsD protein was shown to bind to its own promoter (between arsC and arsD) in addition to the arsR promoter. It appeared that the regulation of expression of the CDB3 ars gene cluster 1 is a very interesting but complicated mechanism

Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1

Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons

Single Molecule Detection in Nanofluidic Digital Array Enables Accurate Measurement of DNA Copy Number

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. The DNA solution is distributed throughout a large number of partitions and then, following amplification, the target DNA copy number in the original solution is estimated based on the proportion of partitions containing amplified DNA. Random distribution of the target DNA molecules and single molecule amplification are critical to the validity of this approach. Here we use a certified plasmid calibrant containing two different target DNA sequences to identify approaches for obtaining the most reliable quantitative data from digital PCR. This fundamental study will increase the confidence of such measurements and advance the use of digital PCR for future bioanalytical research.JRC.D.2-Reference material

A STUDY ON EFFECTS OF MACRO ECONOMIC INDICATORS ON CRUDE OIL PRICES – PHASE TOWARDS BUILDING STRONG MODEL FOR CRUDE OIL PRICE PREDICTION.

Energy is the lifeblood of every economy and a crucial component in its overall success. The economic viability of a project hinges on the fuel supply being reliable, easily available, and environmentally friendly for at least the foreseeable future. For monetary new development import, compelling exploitation of resources and long-stretch acceptance in its utilisation is of paramount significance. Financial indicators allow us to track the health of a country's economy; here, we look at how a rise in the price of crude oil in India's bins as a result of higher prices for crude oil around the world affects other financial metrics, such as the country's Consumer Price Index (CPI) and the value of the Indian rupee. Predictions for the price of crude oil using a univariate analysis approach are also a primary area of interest

PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry

The term ‘gene doping’ is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia

Absolute Quantification of Genetically Modified MON810 Maize (Zea mays L.) by by Digital Polymerase Chain Reaction

Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in realtime PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maizeMON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.JRC.D.2-Reference material

Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction

Abstract Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in realtime PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio