Testing new PCR additives for enhancement of direct PCR -based detection of Y chromosome STRs loci from human hair samples

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Testing new PCR additives for enhancement of direct PCR -based detection of Y chromosome STRs loci from human hair samples
  Testing new PCR additives for enhancement of direct PCR –based detectionof Y chromosome STRs loci from human hair samplesHassab El- Nabi, S. E. 1 ; Elroby, A. M. 2 1 Genetic Engineering and Molecular Biology Division, Department of Zoology, Faculty of Science,Menoufia University, Shebin El- Kom, Menoufia, Egypt2 General Authority of Forensic Evidence, Police Academy, Cairo, Egypt.Corresponding E-mail: ahmedelropy_eg@yahoo.com Abstract: In order to evaluate the effect of adding weak hair detergent on PCR buffer for further direct PCR technique, current investigation was carried out. Hair sampleswere collected from five different individuals and were subjected to direct PCR technique. Two types of commercial detergents and a physical treatment (gentlescratching of outer layer of hair follicle) were added to the PCR buffer. Resultsshowed that Direct PCR over hair samples obtained from five persons whocommonly use hair waxes recorded negative results. When the detergents andthe physical treatment were applied, the scale of peak heights in all loci wassignificantly increased. In addition, results showed that soap foam detergentconsidered the best treatment. Much better results for direct PCR were obtainedafter treating three hairs by these methods, than using only one hair. Thesetreatments may solve the problems of rapid and cost-reducing genotyping of forensic samples as well as improving the detection efficiency for some loci andalso less contaminated DNA templates, especially since most of the convictedsuspects are known to use hair waxes. Key words: Direct PCR, DNA profiles, Forensics, detergents, hair. Introduction: In order to obtain adequate amounts of DNA for downstream applicationssuch as PCR, cloning, and DNA sequencing, DNA extraction is the first step in [1]  many molecular biology experiments ( Sambrook and Russel, 2001 ). Extraction process impose cells to liberate DNA material via physical disturbance and/or chemical processing, which is then followed by a clean-up procedure in whichunwanted cellular components are separated from the DNA ( Hajibabaei et al.,2005 ). During sample extraction, DNA may be lost due to the methods used,which can affect the quality of the DNA profile obtained ( Sawran and Welch,2012 ). Direct amplification has gained increasing interest over the last fewyears. Newer, more robust amplification kits claim to perform directamplification better and faster than kits previously available in the forensicscience community. However, most of the commercially available amplificationkits require pre-treatment of the body fluids with buffers or washing reagents prior to amplification ( Hall and Roy, 2014 ). In 2010, Shokralla et al  . hypothesized that a small amount of DNA oozes from the tissue into the preservation solution (usually ethanol), and that this DNA was amplifiable usinga standard PCR protocol. They considered the preservative ethanol could beused as a source of genetic material for non-invasive analyses or when DNAanalyses are required for specimens that have been consumed in prior exper-iments. They also succeeded to perform their hypothesis and performedamplification to DNA of the agave butterfly larvae, Hypopta agavis that presented in mescal (liquor). A primary advantage of direct amplificationwithout purification of DNA is the reduced analysis time and higher throughputof databank samples ( Hall and Roy, 2014 ). Current methods for detection of short tandem repeat (STR) profiles from reference samples can employ direct polymerase chain reaction (PCR) amplification of body fluids bound to swabs,FTA ®  and/or non-FTA ®  (fast technology for analysis) substrates. Most collectionmedia for storing dried body fluid samples contain cell-lysing chemicals to preserve DNA within a sample that may contain PCR inhibitors ( Burgoyne,1994 ). Concerning human samples, it is currently limited to amplifying bloodand buccal stained FTA ®  cards using specially designed multiplex kits in [2]  forensic DNA. These multiplex kits have improved buffer-polymerase systemswhich are more tolerant to inhibitors present on FTA ®  samples ( Swaran, 2014 ).Furthermore, PPY23 System is configured with the majority of the highlydiscriminating loci with smaller amplicon sizes. Due to smaller amplicon sizeseven, trace amounts of DNA can be amplified and the discrimination potential of  partial profiles can be maximized ( Thompson et al  ., 2013 ). The PPY23 System provides all the necessary materials to amplify the Y-STR regions of humangenomic DNA ( Jain et al  ., 2016 ).In this study, we report a new method for Y-STR detection from hair samples picked from five different individuals using direct PCR after washingsamples with weak detergents. This method kept the desired goal of saving costand time required for obtaining DNA profile as well as having high quality of these DNA profiles. The results were compared on the basis of the scale of peak heights for the generated PCR profiles, between the hair samples that applieddirectly to the PCR only and the same samples that exposed to washingdetergents before entering them to the PCR. Materials and Methods:1-Sample collection and processing: Current study was performed on five volunteers who donated their hairs.Hair samples (1 and 3) were taken from left and right side of the head of each person who commonly use hair waxes. Collected samples were then divided intofour groups. First group was subjected to direct PCR. Hair samples in secondgroup were separately placed (as 1 or 3 hairs for each one) in a 50 mL Falcontube containing 25 mL of soap foam. The hairs were then transferred to another falcon tube containing 25 mL of double distilled water, shaken for washing for 3.5 min, and then this process of washing was repeated twice. The hairs werethen picked, blotted dry in a clean filter paper, and used for direct application toPCR. Third group of hair samples was processed in the same way as the second [3]  group, but without using soap foam and replacing it with 10 mL of detergentmix (1 g sulphonic acid, 20% KOH, 15% sodium lauryl sulfate, 100 mL H 2 O).The 4 th  group of hair samples was only scratched gently for their outer layer of hair root by a razor. In all sample groups no extraction process was applied. 2-Amplification of certain regions on Y chromosome via PCR: PCR amplification of all samples was carried out using PowerPlex ® Y23System (Promega) in a final volume of 13 µL according to (PromegaCorporation Technical Manual # DC2305 and DC2320). PowerPlex ® Y23 kitspecified for certain loci including (DYS19, DYS385a/b, DYS389I/II, DYS390,DYS391, DYS392 and DYS393), DYS438 and DYS439, in addition, DYS481,DYS533, DYS549, DYS458, DYS435, DYS448 and Y-GATA-H4) DYS570,DYS576 and DYS643. Gene Amp PCR System 9700 thermal cycler (AB/LT/Thermo) was used for sample amplification. Amplification set-up andcycling parameters, as described in the PPY23 Technical Manual (PP Y23System Technical Manual, 7/12, available at: http://www.promega.com/resources/protocols/technical-manuals/101/ powerplex-y23-system-protocol/), with consideration of using half the reaction volume of therecommended protocol. Each amplification reaction contained 2.5 μl of PP Y23Master Mix and 1.25 μl of PP Y23 Primer Pair Mix, 0.5 μl of DNA with up to7.75 μl of distilled water. Samples were then initialized for loading in 3500Genetic Analyzer through adding 1 μl of amplified sample or allelic ladder to 10μl of Hi-Di Formamide and 0.5 μl of ILS provided with PPY23 system. Sampleswere denatured for 3 min at 95°C followed by a snap cool in an ice bath. For STR analysis in certain loci, GeneMapper ID v1.5 software (AppliedBiosystems, UK) was used. Negative control was accompanied withamplification. Samples were stored at 4º C for further analysis. Data wasobtained as electropherograms where each locus has one peak except two loci [4]  that may have one or two peaks that termed alleles. The height of peaks isdirectly proportional with the quality of DNA profile.All procedures of sampling followed the Ethics Regulations issued by theResolution of the Egyptian Minister of Health Population No. 238/2003, Articles52-61 and the guidelines from the 4 th  Meeting of the EC International Dialogueon Bioethics (European group on Ethics on Science and New Technologies toEuropean Commission, Copenhagen, Denmark 19 June 2012). All the collectedhairs contained the full hair structure (including the hair root). 3-Statistical data analysis: The Means of the peak heights for analyzed 23 loci were tested for significance by the multiple way analysis of variance (MANOVA) using SPSS statistics 15.0 release 15.0.0 software. Results were considered significant at P ≤0.05. Results: Results presented in table (1 and 2)  and illustrated in figures (1: 6)  showing thedata of Y chromosome profiles of both one and three hairs samples for fiveindividuals who exposed to direct PCR method and three specific treatments toovercome the negative outcome problem of direct PCR method. Amplification products were separated on an Applied Biosystems ®  3500 Genetic Analyzer using a 3kV, 5-second injection as described in the PowerPlex ® Y23 SystemTechnical Manual TMD035. The mean of peak heights for all loci involved in Ychromosome profile and standard deviation errors were obtained where data presented as mean ± standard error of means. Asterisks (   ), refer to a significantdifference between extracted and direct PCR results for the same locus (P<0.05). [5]
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