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  • br Material and methods br Results and

    2020-05-22


    Material and methods
    Results and discussion
    Conclusions
    Acknowledgements S. De thanks DST, New Delhi for generous grant of the SERC Fast Track Scheme No. SR/FT/CS-057/2008. R. Kundu thanks the University of Kalyani for financial assistance. We are grateful to Prof. K. Bhattacharyya of IACS, Kolkata for the time-resolved fluorescence experiments at the set-up under DST project no. IR/I-1-CF-01/02. We acknowledge the help extended by Prof. Tarakdas Basu. S. De and U. Ghosh acknowledge the grants provided from the DST-Purse program, University of Kalyani. We acknowledge CRF, IIT Kharagpur and CSS of IACS, Kolkata.
    Introduction Diamond™ nucleic scid dye (DD) (Promega, Madison, WI, USA) has recently been reported to stain DNA in samples such as hair roots and shafts [1], [2], saliva [3], [4], fingermarks [5] and other forensically relevant items [6]. The DD molecule is an external groove-binder that is able to bind to the backbone of DNA effectively, but it does not bind effectively to RNA and prokaryote supercoiled DNA [1], [7]. DD has an excitation maximum at the blue wavelength (494nm) and emits green fluorescence (558nm) when bound to DNA. The fluorescence signal from DD bound to DNA in hair roots was found to be stable for up to 6 months after staining [8]. A digital microscope “Dino-Lite” (AnMo Electronics Corporation, New Taipei City, Taiwan) is a versatile fluorescence microscope that incorporates suitable excitation illumination and emission GSK2801 for DD and has a wide magnification from 20× to 220×. The microscope receives its power through a USB port of a laptop and the image generated is viewed live on the computer monitor using a simple software application provided with the microscope. Still or video images can be recorded directly to the PC. A dark room is not necessary for collection of a fluorescence signal [5].
    Materials and methods
    Results
    Discussion This described research showed that even when DNA is transferred to a swab and retained for 4 weeks prior to any addition of the stain, fluorescence can still be recorded. This illustrates that the technique is applicable to usage in routine forensic situations where some time delay between collection of samples and DNA profiling of them is experienced. The black cotton swabs are coated by chitosan, which has an antibacterial property [9]. The benefits of this particular feature of these swabs are being investigated further. The effect of 20× DD stained swabs on STR typing through direct PCR amplification resulted in lower RFU values for the two nylon swabs, Cylinder and Ultrafine, compared to the black cotton swab. The black cotton swab generated RFU values comparable to, and no less than, the positive control. It should be noted that the electrical property of chitosan is highly positive at pH 2 and close to neutral at pH 8 [10]. The ideal pH for PCR is about 8.4 at room temperature, which is an optimal for Taq DNA polymerase [11]. Chitosan is a potential medium for DNA collection due to its positive charge in slightly acid solutions. Nylon is a synthetic polymer that also has a slight surface positive charge that is neutralized at a pH of about 5 [12]. Despite this, the amount of PCR amplicon generated from the direct PCR using the nylon swabs (and thus RFU values) are lower than those recorded for the black cotton swabs. The behaviour of nylon and chitosan-coated fibres with regards to collection and retention of DNA is also being investigated further. There was not significant inhibition using nylon swabs as the average RFU results were still higher than 3000 [4]. Previous studies have proved that direct PCR using the nylon swab can generate informative STR profiles [5], [13], [14], [15]. The study looking at the recovery of cellular material showed that there was a higher percentage of recovery of DNA as a result of swabbing a non-stained fingermark than when applying DD to the fingermark and then collecting with a swab. DD, which is an external binder, binds to the sugar phosphate backbone of DNA, which is negatively charged [7]. It is therefore feasible that the overall negative charge of DNA molecules will be altered by staining, thus reducing the binding between DNA and fibres of swab.