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Removal of Contaminating Genomic DNA in QRT-PCR using a Shrimp Nuclease
Nicky Quipse, Saima Naveed Nayab and Ian Kavanagh
DNA contamination can often occur in quantitative reverse transcription – polymerase chain reactions (QRT-PCR), and should be removed in order to avoid false positive results. DNase I is commonly used for removing DNA contamination, but this has a relatively long and harsh protocol which introduces an extra step between the isolation of RNA and the QRT-PCR reaction itself, as well as increasing the risk of RNA degradation due to the harsh inactivation conditions.An endonuclease from arctic shrimp Pandalus borealis has properties that make it useful for the removal of contaminating DNA. The endonuclease activity of the enzyme is specific to double stranded DNA, which therefore allows the enzyme to be added directly into the reverse transcription step. Unlike DNase I, the shrimp nuclease is easily inactivated at high temperatures, such as those used for the RT deactivation / hot start incubation step of a QRT-PCR reaction, and therefore can be used to selectively degrade double stranded DNA, leaving single stranded DNA and RNA intact.
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The Effect of Amplicon Charcteristics on the success of Fast QPCR
Gerwyn Jones, Sryana Kapavaparu, Saima Nayab and Ian Kavanagh
Employing fast QPCR cycling protocols is a simple and effective way of maximising throughput by reducing run durations. Fast cycling protocols can easily be achieved by reducing the temperature step dwell times.It has been reported that the use of fast QPCR protocols can result in a loss of sensitivity and greater inter-replicate variance with some assays. It is therefore important to realise that speeding up cycling protocols can be detrimental to the quality of results, but not in all cases. The factors which determine whether an assay can be successfully employed using fast cycling conditions are poorly understood.
The effect of three target amplicon characteristics on the success of fast QPCR was investigated using a panel of characterised assays. The amplicon characteristics focused on in particular were base-pair length, GC content and minimum ΔG at 60°C, the latter being a measure of the severity of amplicon secondary structure at the annealing temperature.
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The Effect of White Pigmentation in 96 Well Plates during QPCR
Saima N. Nayab, Philip N.Harries, Simon C. Baker and Ian Kavanagh
QPCR instruments are able to monitor amplicon quantity in real-time during PCR reactions by detecting fluorescence signals and recording fluorescence data. The majority of fluorescence is reflected out of the QPCR plate either by the polypropylene itself or by the walls of the thermal cycler block, when natural polypropylene is used. QPCR plates containing a white pigment improve reflection towards the fluorescence detector inside the thermocycler during a QPCR run (Figure 1 and Figure 2).more...