Agarose gel electrophoresis is a common molecular biology technique used to separate DNA or RNA molecules by size. This is achieved by denaturing and applying a negative charge to our nucleic acid sample. The samples are then run through an agarose matrix with an electric field (Electrophoresis). Shorter molecules move faster and thus migrate further and in the gel over a given time period. Many factors are involved with regards to the final visualization step and achieving high resolution of the resulting bands on the agarose gel.
Tips to help improve resolution include: - Running the gel at a lower voltage for a longer period of time - Using a wider/thinner gel comb - Loading less DNA into each well
MangoTaq comes with a coloured reaction buffer that contains red and orange dyes, which separate during electrophoresis and provide quick reference points for monitoring the mobility of the DNA samples in the gel.
It's time for the second in our series of Bioline Scholar Monthly compilations. This month we're focusing on the many uses of Bioline polymerases in forensic science.
Identification of a report's species is one of the basic analyses in forensics. However, due to the nature of the sampling environment, DNA samples often contain PCR-inhibitory substances which may generate blank or incomplete DNA profiles. The common approach to overcoming PCR inhibition is extensive DNA purification, but this can increase the risk of DNA loss. In some cases, isolation of single cells using laser-capture microdissection can be used, but again this reduces the amount of DNA available.
DNA polymerases that can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition, without any additional sample preparation, are therefore advantageous, as are polymerases that result in high yields.
Bioline’s DNA polymerases are very robust and have been carefully designed to overcome these problems. BIO-X-ACT™ Short in particular is specifically designed for difficult/problematic PCR applications that require high processivity and fidelity, applications that would normally fail with other DNA polymerase. MangoTaq™ has also been designed for problematic and ancient DNA, whereas IMMOLASE™ and BIOTAQ™ are high yield for small sample sizes. Together with our new MyTaq™, MyFi™ and RANGER Bioline has polymerases to meet all the high-fidelity requirements of forensic science.
So, without further ado, here's...
Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In Barkway and colleagues procedure, BIO-X-ACT Short DNA Polymerase was initially used to successfully verify the LAMP primer pair for Eimeria species specificity using PCR.
Barkway, C. P., et al. BMC Veterinary Research 2011, 7:67 (2011) – Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens
In a comparison of nine DNA polymerases, the DNA detection limit was lowest with BIO-X-ACT Short, providing the highest number of improved DNA profiles, using real crime scene saliva samples.
Hedman, J., et al. BioTechniques 47, 951-958 (2009) – Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles
The identification via DNA analysis is reliably and reproducibly possible from well preserved and semi-burnt bones.
Schwark, T., et al Forensic Sci. Int.: Gene. 5(5), 393-399 (2011) – Reliable genetic identification of burnt human remains.
Dried herbarium specimens may be invaluable to understand long-term changes at sites with a history of cyanobacterial blooms.
Metcalf, J.S., et al Harmful Algae 15 47–52 - (2012) – Analysis of microcystins and microcystin genes in 60–170-year-old dried herbarium specimens of cyanobacteria
Out of 19 polymerases, the best performance was exhibited by the Mango-Taq DNA polymerase, which was the only polymerase which was able to amplify the ~620 bp amplification product from the 102 year old sample.
Telle, S. & Marco Thines, M. PLoS ONE 3(10):doi:10.1371/journal.pone.0003584 - (2008) – Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases
In Australia and globally, Sarcophagidae flies remain unexploited as indicators of post-mortem interval in forensic investigations. A molecular identification method involving DNA ‘barcoding’ of the mitochondrial COI gene from 16 species of Australian Sarcophagidae was successfully developed. The authors conclude analysis of sarcophagids in forensic entomology should increase and their value as tools in criminal investigations realised.
Meiklejohn, K. A., et al. Int. J. Legal Med. 125(1), 27-32 - (2011) – DNA-based identification of forensically important Australian Sarcophagidae (Diptera)
Microsatellite markers were developed for the medicinal plant Tripterygium (Celastraceae) to assess its population structure and to facilitate source tracking of plant materials used for medicinal extracts.
Novy, A. & Jones, K. C. Am. J. Bot. 98(10) e280-e281 (2011) – Characterization of polymorphic microsatellites for Tripterygium (Celastraceae), a monospecific genus of medicinal importance
Lindgren funnel traps baited with aggregation pheromones are effective tools for monitoring flight activity in the red flour beetle (Tribolium castaneum) and lesser grain borer (Rhyzopertha dominica). But the samples are affected by the preservation method.
Stevens, M. M,. et al. J. Stored Products Res. 47(2), 69–75 - (2011) – Maintaining DNA quality in stored-grain beetles caught in Lindgren funnel traps