Optimization of DNA concentration to amplify short tandem repeats of human genomic DNA

Analysis of the length polymorphisms of short tandem repeats (STR) loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. Th e purpose of this study is to optimize the DNA concentration in ng/μL for amplifi cation of DNA markers. AmpFlSTR Identifi ler Kit is used to amplify STR markers and capillary electrophoresis is used to analyze DNA profi le of human the genome. Two sets of samples with following DNA concentration:  pg –  ng/ μL were used for this study. Th ere was no DNA profi le detected in samples with concentrations  pg  pg/  μL (pictogram), while in some cases partial DNA profi le was yielded. On the other hand samples with . ng –  ng/  μL, yielded a full DNA profi le. We were not able to obtain any profi le using concentrations over  ng/  μL. Improvements in detection limits/sensitivity at upper and lower DNA concentrations are of potential benefi ts to amplify STR of Human Genomic in order to obtain a full DNA profi le. Th e optimal DNA concentrations which produced reliable and balanced peaks, no off scale peaks and full DNA profi le for all loci were at range . ng –  ng/  μL. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Eukaryotic chromosomal DNA, polymorphic short tandem repeat (STR) loci are key tools for: rapid gene discovery, disease locus mapping and carrier diagnosis of disease states, linkage analyses, agricultural genetics, parentage assessment, and population diversity studies.Th e ability to detect genetic differences between individuals increases when DNA typing information at multiple polymorphic STR loci is combined [, ].Clinical diagnostic laboratories usually perform analyses of biological samples that have been collected and stored in ideal conditions, making sample quality and quantity rarely an issue.Th ose ideal conditions are usually not met in the DNA typing analysis of forensic biological evidence, as there is no control over the amount of biological material left at a crime scene or its level of degradation or contamination due to exposure to various en-vironmental insults [, ].Forensic biological samples may present challenges for DNA typing analysis, even with the utmost care throughout crime scene evidence recovery and storage.Due to potential exposure to a virtually unlimited number of uncontrolled variables, forensic casework specimens may result in particularly challenging polymerase chain reaction (PCR) templates.Optimized concentration of DNA and reagents is very important on PCR amplifi cation of different combinations of  polymorphic tetranucleotide STR loci in a single reaction tube.Th is study has done to check the upper and lower limits of DNA concentration used in amplification step to obtain full DNA profiles [].

Samples
Our study was performed using human blood as biological material.Before analysis the blood sample has dried on fi lter paper.Th e size of the samples used for extraction were x mm  and Chelex- resin is used as extraction method [].[], [].Th e amplifi ed DNA markers were: DS, DS, DS, CSFP, DS, TH, DS, DS, DS, DS, vWA, TPOX, DS, Amelogenin, DS and FGA [], [].Finally detection of amplified samples was done using . μl of amplifi ed DNA product that was combined with aliquot  μL of the Hi-Di™ Formamide (AB P/N ) mixed with GeneScan™  LIZ™ Size Standard (AB P/N ).Capillary electrophoresis was carried out using an automated DNA sequencer ABI PRISM®  Genetic Analyzer, AB P/N --/-W.Th e raw data were analyzed using GeneMapper® ID Software v..The minimum peak height threshold was set at  RFU [, ].DNA concentration between  - ng/ μL yielded full DNA profiles but there were allelic imbalance peaks and signifi cant diff erence on RFU in heterozygote peak height ratios between the shorter and longer loci (Figure ).

Samples
For shorter loci RFU was over  to , whereas for longer ones were under .Despite these results, DNA profile obtained from above samples can be used officially as DNA Profile at System of Justice.No DNA profile was yielded in samples over  ng/μL (Figure ).Some loci were obtained, but just few of them (below ) so they did not represent any relevance in DNA profi le.To be valid the DNA profile must have at least  loci obtained.Those profiles with number of loci below  are unavailable to be used for any purpose [], [].The referent samples yielded a full DNA profi le.Th e best DNA profi le was obtained from samples with DNA concentration at . -. ng/ μL.Reliable DNA profiles were obtained from samples with ., . and  - ng/ μL as well (Table.).

DISCUSSION
A variety of commercial Kits for DNA amplification are now available and they routinely are used in forensic and diagnostic laboratories worldwide.Although generally reliable and convenient, many of these Kits offer only a relatively limited dynamic range for upper and lower limits, so that in many cases samples from the crime scene, reference samples and clinical samples frequently have to be diluted or concentrated and retested in order to avoid exceedi ng the upper or lower limits.At the same time analyzes is performed by using real-time PCR which is time and money consuming.Th e advantage of the current study is the fact that we found that upper and lower limits beyond of what is actually suggested by companies.Th is method increases the opportunity to amplify DNA directly from extraction step into amplifi cation

CONCLUSIONS
AmpFlSTR Identifiler, the PCR Amplification Kit used to amplify STR markers of analyzed samples exhibited to be very stable.Primers of this Kit were sensitive and very specifi c for DNA markers as mentioned above.Even if AmpFl-STR Identifi ler PCR Amplifi cation Kit -User manual suggests using DNA concentration . -. ng/ μL (lower and upper limits) [], and National Institute of Standards and Technology (NIST) suggests . - ng/ μL as the best range of STR Kits [].We have obtained full DNA profi le with a lower concentration of . ng/ μL up to  ng/ μL for the upper one.Other reagents of this Kit: AmpFlSTR® PCR Reaction Mix, AmpliTaq Gold® DNA Polymerase and AmpFlSTR® Identifiler™ Allelic Ladder used on μL as a master mix shown to be enough for amplification of samples with concentration  ng/μL without any problem for inhibition by the template.Performing the step of DNA quantifi cation on RT-PCR is time consuming and cost eff ective.We found that the best and reliable results are yielded beyond the limits suggested by different companies.We obtained reliable DNA profi le from concentration up to  ng per sample analyzed.Using those results, the laboratories can make validation of methods in different applications such as: molecular biology, genetics, biomedicine etc to analyze samples directly from extraction to amplifi cation and by skipping quantifi cation step in RT-PCR.By applying such approach it could be possible to save time and money.
with DNA concentration at . and . ng/ μL yielded no DNA profi le (Figure).No DNA profi le was obtained from samples with . ng/μL, however in this case some loci were included (Figure).Baseline had noisy and allelic imbalance as well.Samples with DNA concentration from . - ng/μL yielded full DNA profiles (Figure).Th ere were no any allelic imbalance, stutter or any other artifacts except for the sample concentration at  ng/μL.Samples with concentrations . and  ng/μL exhibit decrease of Relative Fluorescence Units (RFU) starting from shorter loci toward longer ones for all panels.Th e loci with lower RFU were DS, CSFPO, DS, DS and FGA[].

FIGURE 2 .
FIGURE 2. Presents electropherogram of samples with Partial DNA Profi le analyzed with AmpFlSTR Identifi ler Kit.There are three alleles obtained in total of 16 human genome loci analyzed.

FIGURE 1 .
FIGURE 1. Presents electropherogram of sample with No DNA Profi le analyzed with AmpFlSTR Identifi ler Kit.There is no any allele obtained in total of 16 human genome loci analyzed.

FIGURE 4 .
FIGURE 4. Presents electropherogram of samples with Full DNA Profi le analyzed with AmpFlSTR Identifi ler Kit.There are all alleles obtained in total of 16 human genome loci analyzed.At some alleles there are some imbalance peaks.

FIGURE 3 .
FIGURE 3. Presents electropherogram of samples with Full DNA Profi le analyzed with AmpFlSTR Identifi ler Kit.There are all alleles obtained in total of 16 human genome loci analyzed.
 cycles of  o C for  min,  o C for  min and  o C for  min,  o C extension for  min and - o C until samples were analyzed further.Th e amplifi cation was carried out on Ge-neAmp PCR System  (AB P/N N-)

TABLE 1 .
Represents DNA profi le yielded on diff erent DNA concentrations one without any trouble toward result interpretation.DNA yielded profi le from an amount up to  ng/ μL (which is a double amount suggested by protocols), crucial due to the fact that is easy to determine that approximate amount in our sample size during the extraction step.Th is decreases a workfl ow of our analysis which in this case will consist of extraction, amplifi cation and detection without quantifi cation step.This workflow would not have any interference in terms of detecting sensitivity of DNA.In addition, this method can serve as a great tool to help laboratories in obtaining reliable DNA profiles by saving time and money.Leclair et al.have studied lower and upper limits for DNA concentrations and total volume used for DNA amplification.In this study the best results were yielded using . ng of DNA in a -μL PCR reaction volume which appeared to produce an identical profile to  ng of the same DNA in a -μL reaction volume[].Company Applied Biosystems which has performed a validation of the AmpFlSTR Identifi ler Kit, suggested using an amount of DNA concentration in the range of . - ng/ μL[].