Optimisation of Forensic Genetics Procedures Used in Disputed Paternity Testing : Adjustment of the PCR Reaction Volume

Standard molecular techniques, with only a slight modifi cation, are very useful in obtaining and interpreting the fi nal results in the fi eld of forensic genetic. Data obtained through such analysis are highly reliable and can be used as a very powerful tool that produces valuable results. However, success and swiftness of DNA typing of biological evidence either that found at a crime scene or used in disputed paternity testing, depends on the optimization of numerous factors. One of the most important and critical phases that ensures reliability of the whole procedure is the choice of the most suitable volume for the amplifi cation protocol. Buccal swabs were collected from volunteers. DNA was extracted by Qiagen DnaeasyTM Tissue Kit (Qiagen Co. ). PowerPlex  kit (Promega Corp., Madison, WI) was used to simultaneously amplify  STR loci by PCR. Amplifi cation was carried out as described previously (Promega Corp. ). Th e tested total working reaction volumes were ,  and μl. Th e PCR amplifi cation was carried out in PE Gene Amp PCR System Th ermal Cycler (ABI, Foster City, CA). Amplifi cation products were analyzed on an ABI PRISM  instrument (ABI, Foster City, CA) in  bis-acrilamide gel. Amplifi cation was generally successful for all the tested reaction volumes. Lower partial to complete DNA profi les ratio, the quality of obtained STR profi les, signifi cantly reduced amount of reaction’s components give advantage to μl reaction volume over other two tested vol-


Introduction
Forensic genetics greatly benefi ted from the discovery of technique known as the polymerase chain reaction (PCR).Th is neat chemical reaction, fi rst described in  by Kary Mullis, has revolutionized molecular biology ().PCR is an enzymatic process in which specifi c regions of DNA are duplicated repeatedly to yield millions of copies of a particular sequence in a matter of few hours (,).Th e procedure involves heating and cooling of chemical mixtures in a precise thermal cycling pattern.Hypothetically, approximately billion copies of the target DNA region are generated after  cycles.PCR reaction is a mixture of several components dissolved in DNA-free deionized water to reach preferred concentration of each constituent.Th e fi nal component is the DNA template.Th e PCR process consists of three steps that are cyclically repeated.First, DNA strands are separated by heating (denaturation).Second, primers (DNA oligonucleotides) hybridize with target sites of these single stranded molecules to defi ne the sequences to be amplifi ed.Each primer performs as a starting point for the third step -the duplication of the target sequence.Th is last step occurs in the presence of the enzyme DNA polymerase and four nucleotide building blocks ().STR loci, commonly used today in human DNA typing are usually assembled within one of commercial STR multiplexes.Th e availability of these STR kits that permit amplification of more than  STR loci has truly revolutionized forensic DNA analysis.Incredibly low matching probability (less than one in a billion) and possibility of successful amplifi cation with only ng of DNA template promote these kits as powerful tools for various DNA testing applications ().Also, the kits have signifi cantly simplifi ed preparation of PCR reaction.According to the manufacturers' recommendations, DNA templates just need to be added to a pre-made PCR mix containing all the necessary components for the amplifi cation reactions.Th ese kits are usually highly optimized by manufacturers.Any additional optimization is usually carried out by diff erent user laboratories, according to their needs and purposes.An extremely important feature in the advanced DNA paternity testing, besides extremely high probability of paternity (>,), is swiftness and simplicity of the whole method.Nowadays, as a result of intense optimization of the DNA isolation, amplifi cation and detection procedures, the entire process can be performed within - hours.Hence, this type of testing is promoted to almost a standard practice in modern society.Our preliminary review represents small, but for our laboratory very valuable, contribution to optimization of forensic genetics procedures used in disputed paternity testing.

SAMPLES COLLECTION
Six volunteers duly informed about the research donated DNA samples.Th eir buccal swabs were air-dried, sealed in , ml tubes, and immediately transported to the Laboratory for Forensic Genetics at the Institute for Genetic Engineering and Biotechnology, Sarajevo, B&H.Th e samples were stored at -oC until DNA was extracted.
DNA ANALYSIS Qiagen Dnaeasy TM Tissue Kit was used for DNA extraction according to the manufacturer's protocol ().Pow-erPlex® System (Promega Corp., Madison, WI) was used to simultaneously PCR amplify  autosomal STR loci and amelogenine.Th e STR loci are: DS, TH, DS, DS, Penta E, DS, DS, DS, DS, CSFPO, Penta D, vWA, DS, TPOX and FGA.Amplifi cation was carried out as described previously ()

Results
Th e characteristics of all the obtained STR profi les are presented in Table .Th e number of detected loci varies from eight to sixteen per generated PP STR profile.The weakest PP STR profile was detected for Sample  at  μl reaction volume.Only eight STR loci were detected for this reaction volume.Th e highest variation was observed within samples amplifi ed in  μl reaction volumes.On the contrary, the highest fidelity was achieved with samples processed in  μl total volume -all the generated profi les were complete ( loci).Means and variability measures (based on peak height) for the three tested volumes are showed in Table  and Table 

Discussion
Application of STR loci in the interpretation of genetic profiles from different biological sources may be extremely valuable not only in a number of investigative procedures (routine paternity testing, forensics and mass disaster human identifi cation), but also in diff erent population studies ().Numerous previous studies (, , ) describe PowerPlex® System (Promega Corp., Madison, WI) as a very powerful tool that produces valuable results.Previous Bosnian experiences confi rm this (-).Th erefore, we chose this STR multiplex system as a principal method for DNA typing performed in our laboratory.PCR is commonly performed in reaction volume range of - μl ().Evaporation and accurate pipetting may cause a problem, or at least a challenge, with low volumes reaction.Conversely, larger reaction volumes encounter a problem of even heating and cooling throughout the entire volume.Th erefore, in most molecular biology protocols PCR reaction volume is maintained in - μl range ().According to the results of previous studies, manufacturer of PowerPlex® System (Promega Corporation) suggested total reaction volume of  μl, which was accepted and applied in earlier studies (,).Th us, in our earlier work we followed the same pattern ().However, of recently, we have successfully used μl as the total reaction volume (-).Since the type of biological sample used in these studies was the same type that we usually employ in routine disputed paternity testing, we have decided to compare three diff erent PCR total reaction volumes and select the one that suits our procedures best.Analyzing the number of the obtained STR loci for each sample we observed that the samples processed in  μl reaction volume resulted in complete profi le.Th e fraction of detected loci per profi le diff ers in other two tested volumes ( and  μl).It is important to emphasize that RFU threshold was set at  units, and all peaks below the threshold, regardless whether they were detectable/ useful or not were discarded (which was the case with most of the loci described as undetectable).Furthermore, calculated variability measures for diff erent total reaction volumes of each sample, as well as the same parameters calculated for joint results clearly indicate that  μl volume signifi cantly diff ers from  μl and  μl.Th e most illustrative is variability coeffi cient, which is signifi cantly lower within  μl then  and  μl group.Th at means that the peak heights between diff erent loci are better balanced and that the profi les obtained in  μl reaction volume are more transparent than those prepared in the other two volumes.Results obtained in this preliminary study suggest that, due to better ratio between the obtained complete and partial DNA profi les, quality of the obtained STR profi les and signifi cantly reduced amount of reaction components,  μl total reaction volume is more suitable for using in these particular settings.Th ose would be our guidelines in our further examination that should include larger number of the examined samples and more relevant parameters (i.e.occurrence of stutter-peaks, balance of peak area etc.).
in total reaction volumes of ,  and  μl.Th e PCR amplifi cation was carried out in PE Gene Amp PCR System Th ermal Cycler (ABI, Foster City, CA) according to the manufacturer's recommendations:  o C ( minutes),  o C ( minute),  cycles of: ramp  to  o C for  seconds, ramp  to  o C for  seconds and ramp  to  o C for  seconds,  cycles of: ramp  to  o C for  seconds, ramp  to  o C for  seconds and ramp  to  o C for  seconds,  o C ( minutes) and  o C maintain.Amplification products were separated on an ABI PRISM  instrument (ABI, Foster City, CA) in  bis-acrilamide gel (Long Ranger® Single® Packs).Raw data were compiled and analyzed using the accessory software: ABI PRISM® Data Collection Software and Gene Scan®.Numerical allele designations of the profi les were obtained by processing with Powertyper TM  Macro.Th e threshold for the sample analysis was set at  rfu (relative fl uorescence units).
. Table  represents the measures obtained for each individual sample while Table  represents the same parameters based on joint results for each tested reaction volume.The calculated statistical parameters based on individual sample results as well as joint results clearly indicate that, at the , significance level, the means of  μl group is significantly diff erent then other two ( μl and  μl) groups.
DAMIR MARJANOVIĆ ET AL.: OPTIMISATION OF FORENSIC GENETICS PROCEDURES USED IN DISPUTED PATERNITY TESTING: ADJUSTMENT OF THE PCR REACTION VOLUME DAMIR MARJANOVIĆ ET AL.: OPTIMISATION OF FORENSIC GENETICS PROCEDURES USED IN DISPUTED PATERNITY TESTING: ADJUSTMENT OF THE PCR REACTION VOLUME