Summary: The induction of drug metabolizing enzymes can narrow therapeutic windows by reducing or eliminating the effectiveness of pharmacological agents. After observing that an increase in total bilirubin level correlated with faster systemic clearance of an aromatase inhibitor agent, the authors used RT-PCR to analyze the expression of known drug metabolic enzymes in bilirubin-treated hepatocytes. Transcripts analyzed included nine different cytochrome P450 (CYP) genes, seven UDP-glucuronosyltransferase (UGT) genes, constitutive androstane receptor (CAR), pregnane X receptor (PXR), retinoid X receptor α (RXRα) and hepatocyte nuclear factor-4α (HNF-4α). PrimeSTAR Max DNA Polymerase was used during RT-PCR with an extension step of 5 seconds at 72°C.
Summary: The human NOTCH1 gene, which controls development, proliferation, differentiation, and survival of T-cell progenitors, is encoded by a 50-kb locus spanning 34 exons at chromosome 9q34.3. An extremely rare but recurrent translocation had previously been identified in this region that was associated with T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL). This transposition juxtaposes NOTCH1 with the T-cell receptor b locus (TRB@) at 7q34, leading to over-expression of a truncated form of NOTCH1 protein under control of TRB@ regulatory elements. In this study, a 41-year-old patient with T-LBL was found to have a novel TRB@/NOTCH1 fusion gene with a NOTCH1 breakpoint in exon 25. The consequence of this novel fusion was overexpression of truncated NOTCH1. The authors postulate that ligand-independent, constitutive activation of the NOTCH1 pathway occurred, and speculated that therapy with gamma-secretase inhibitors may be effective in such instances. PrimeSTAR Max DNA Polymerase was used for PCR amplification of 3.3 and 3.9 kb fusion products from human genomic DNA with a 30-second extension step.
Summary: In a proof-of-concept study aimed at rapid detection of viral pathogens, researchers developed a microfluidic device to detect AH1pdm influenza originating from swine. The device included a microfluidic chip on which RT-PCR was performed by circulating the reaction between different temperature regions. Products were detected with a disposable electrical printed (DEP) chip, and Methylene Blue (MB) was used as the reporter for electrochemical detection of the amplicon. The enzyme and reagent mix included PrimeScript Reverse Transcriptase, SpeedSTAR HS polymerase, PrimeSTAR Max polymerase, RNase Inhibitor, dNTPs, primers, BSA (to prevent adsorption of the enzymes to the polydimethylsiloxane (PDMS) microfluidic chip), and isolated AH1pdm virus RNA. Total flow time for RT-PCR was 15 minutes with product amplification observed over 9.5 minutes.
Summary: Focusing on hand, foot, and mouth disease—a serious public health issue in China with over 7.2 million cases reported from 2008-2012 and 2,457 deaths—scientists reported a proof-of-concept study using a capillary convective PCR (CCPCR) device for RT-PCR. The closed-system device included an amplification tube housing RT and PCR reactions and a dipstick assay component for visualizing PCR products. Enzymes and reagents including SpeedSTAR HS polymerase were lyophilized and sealed in the device, preventing the need for refrigeration (an important consideration for point-of-care applications). Running the reaction involved adding DEPC water to the device to reconstitute the reagents and adding RNA isolated from clinical samples (throat swabs). The bottom of the device was heated at 58°C for RT (5 min.), then at 95°C for PCR (20 min.). The 95°C incubation established a convective temperature gradient through which reagents circulated spontaneously, undergoing the three steps of the PCR cycle as they flowed through the corresponding temperature zones. Sample was then wicked into the detection dipstick for visual evaluation of experimental and control lines. The devices were used to test 141 clinical samples. The authors reported sensitivity and specificity of 98.5% and 100%, respectively.
Summary: With a goal of improving the sensitivity and speed of microfluidic PCR, the authors developed a method for segment-flow PCR in a microfluidic device. To reduce reagent depletion they performed pre-treatment of the microchamber with PCR reagents. Amplification of uidA from E. coli DNA was used as a test assay. The authors reported product detection within 8 minutes (15-fold faster than conventional PCR methods) at a sensitivity equivalent to four E. coli cells per µl (equivalent sensitivity to conventional PCR). SpeedSTAR HS polymerase was used in this study.
Summary: The authors developed a microfluidic device and methods capable of direct PCR from saliva samples. PCR assays were conducted directly on saliva samples spiked with a dilution series of CMV viral DNA or sex determining region Y (SRY) DNA. Products were detected in situ with an optical detection component. SpeedSTAR HS polymerase was used, and 35 cycles of amplification were completed in 25 minutes.
Summary: In an effort to speed up protocols for the multiplex amplification of STR loci, researchers with the Royal Canadian Mounted Police used SpeedSTAR HS polymerase with the AmpFlSTR Profiler Plus assay. The authors wrote, “By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR HS) and a more efficient thermal cycler instrument (Bio-RAD C1000T), we were able to reduce the amplification process from 4 h to 26 min.” SpeedSTAR HS enzyme was compared to polymerases offered by several other competitors. The authors wrote, “When the two ‘fast’ polymerases were used in conjunction with their respective amplification buffers, only the SpeedSTAR HS DNA polymerase was able to produce amplified products at all loci in the specific conditions tested.” The assay was tested with 151 forensically relevant samples such as blood stains on fabric, wood, glass, or cement, buccal swabs, vaginal swabs, semen samples, chewing gum, hair samples, fingernail clippings, cigarette butts, and a food sample (pizza dough).
Summary: To achieve ultra-fast PCR, researchers developed a thermal cycling system that utilizes convective fluid flow through a porous media matrix. The system was designed to enhance heat transfer rates, and was used with SpeedSTAR HS polymerase or an enzyme from another manufacturer to amplify targets from Erwinia herbicola DNA or synthetic DNA from the SARS virus. Total reaction times of under three minutes were accomplished.
Summary: Using a disc-type reaction vessel, specialized PCR thermal cycler, and SpeedSTAR HS polymerase, the authors developed methods for high-speed PCR (Hyper-PCR) with total reaction time of 11 minutes. Clinical samples (147 specimens including throat swabs, fecal samples, nasal swabs, cerebrospinal fluid samples, and a conjunctival sample) were tested for Adenoviral (Ad) DNA using this system. In comparison to a conventional real-time PCR assay as a gold standard, the Hyper-PCR assay was reported to have 100% sensitivity and 100% specificity.