|On chip micro-extraction and real-time PCR with integrated SPAD optical fluorescence detection for nucleic acid analysis|
Cristina Potrich, Elisa Morganti, Nicola Massari, Lucio Pancher, C. Kostoulas, Laura Pasquardini, Cristian Collini, Andrea Adami, Lorenzo Lunelli, F. Kalatzis, David Stoppa, Cecilia Pederzolli, Leandro Lorenzelli
A PDMS lab-on-a-chip for one step DNA isolation and real time-polymerase chain reaction (RT-PCR) has been designed, fabricated, and characterized for point-of-care clinical diagnostics. In addition, a module for on-chip optical detection based on SPAD - Single-Photon Avalanche Diode - detector has also been developed and used to monitor the presence of specific DNA polymorphisms possibly related to genetic diseases.
|Chemical Variant of 7-deaza-dGTP for Improved CG-rich PCR Amplification|
E. H. Ashrafi, S. Shore, T. Le, V. Timoshchuk, N. Paul, R. Hogrefe, G. Zon, I. Koukhareva, A. Lebedev
PCR amplification of nucleic acids is a fundamental technique used in many molecular biology laboratories. Despite its wide use, certain GC-rich regions of DNA, such as mycobacterial disease targets, still remain a challenge for amplification. Sequences high in GC content are associated with the formation of secondary structure, which prevents adequate strand separation and DNA polymerase amplification. As a consequence, mispriming is prominent, complicating specific product formation.
|Periodontal Pockets Microbiota qPCR Analysis at Different Stages of Periodontitis|
Oksana Zorina and Denis Rebrikov
Six major periodontal pathogens were quantified (with standardization to human gDNA) in periodontal pockets of periodontitis patients and healthy subjects by original qPCR assay. Relative amount of pathogens to total bacterial mass increased for P.gingivalis (100-fold), P.intermedia (10-fold) and T.forsythensis (10-fold).
|Low-volume on-chip single sperm cell analysis|
Pickrahn I. E. ,Schmidt-Gann G., Kroneis T.
We performed low-volume on-chip DNA typing of single sperm cells isolated by means of laser microdissection. 16plex PCR was successful in 39 of 40 single cell samples yielding a mean PCR efficiency of 62.8%. In addition, we were able to identify a single-cell sample containing more than one cell enabling us to monitor the quality of the whole procedure, and hence, exclude “contaminated” samples from further analysis.
|Development and NDA-Level Validation of a Real-Time PCR Procedure for Detection and Quantification of Residual E.coli DNA Contamination of Biopharmaceutical Products|
Dan Papa, Pedro J. Morales and Michael D. Sadick
Escherichia coli (E. coli) has been commonly used for the production of biopharmaceuticals. Among the impurities that must be monitored in biopharmaceuticals is residual host-cell DNA (HCD). This study describes the development and subsequent NDA-level validation of a real time PCR procedure developed in response to a client’s need to improve the sensitivity of detection and quantification of residual E. coli HCD in their drug product.
|Chemically Modified Primers for PCR and Ligation Applications|
Elena Hidalgo Ashrafi, Sabrina Shore, Tony Le, Victor Timoshchuk, Natasha Paul, Richard Hogrefe, Inna Koukhareva, Alexandre Lebedev
PCR is an essential tool with utility in a variety of advanced applications. To improve the specificity of PCR, a unique approach to "Hot Start" PCR employing primers containing thermolabile modifications has been developed. These modified primers, named CleanAmp™ Primers, are amenable for use in Hot Start activation schemes as the modification is released after an initial denaturation step.
|Development of a Test Battery for Epigenetic Non-genotoxic Carcinogens|
The main objective of this sudy is to obtain an increased insight into the mechanisms of action of epigenetic carcinogens. Although the expected number of non-genotoxic carcinogens among newly registered compounds is unknown, there is a growing concern that when numbers of 2-year cancer bioassays are significantly reduced, non-genotoxic carcinogens may go undetected. Therefore there is a need for the development of alternative methods for their detection.
|Chemically Modified Primers for Improved Multiplex PCR|
Elena Hidalgo Ashrafi, Tony Le, Alexandre Lebedev, Richard Hogrefe, Victor Timoshchuk, Sabrina Shore, Inna Koukhareva and Natasha Paul
Multiplex PCR is an advantageous technique used in PCR applications to amplify multiple targets in a single reaction. As useful as it is, this technique presents a new set of challenges that further complicates PCR setup. For example, reactions are more prone to off-target amplifications such as mis-priming and primer dimer due to the increased number of primer pairs.
|Hot Start dNTPs - Novel Chemistries for Use in Advanced PCR Applications|
Tony Le, Elena Hidalgo Ashrafi, Sabrina Shore, Victor Timoshchuk, Natasha Paul, Richard Hogrefe, Inna Koukhareva, Alexandre Lebedev
PCR is a widely used scientific tool whose specificity can be increased by the use of Hot Start technologies. Although many Hot Start technologies exist, recently developed CleanAmp™ dNTPs are a distinct approach that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3´-hydroxyl.