Utilizing Short Tandem Repeats (STRs) as a Resolving Matrix in Parental Dispute DNA Analysis ()
1. Introduction
The modifications in inherited regions of the DNA from one individual to another which are described as polymorphic regions are very important in forensic cases and paternity disputes. Short tandem repeats which are sub groups of variable number tandem repeats (VNTR) loci are highly abundant in human genome. Usually while measuring in lengths of 2 - 7 pairs which are repeated in head-tail manner, short tandem repeat polymorphism are attributed to the different number of copies of the repeat elements that can occur in a population of individuals [1] [2] . Before using a four locus multiplex short tandem repeat (STR) model into case work, an extensive series of tests were carried out to determine and validate valuable procedures for assessing the evidential proof of a match between crime and suspect scene [3] [4] [5] [6] . Deriving from case work which were analysed from three main ethnic groups earlier encountered in the United Kingdom twelve data bases which were analysed. These ethnic groups include Asians from the Indian sub-region, Caucasian and Afro-Caribbean. Results from these ethnic groups gave credence for the need to investigate the impact it will have on forensic case work [7] [8] [9] [10] . Despite the enormity and quantum of STR contained in the human genome, a very few core set of loci have been selected for use in forensic DNA and human identity testing [11] [12] [13] . Moreover, core loci permit equivalent genetic information to be shared and compared. A detailed process for short tandem repeat typing entails sample collection, DNA extraction, DNA quantitation, PCR amplification of multiplex STR loci allele separation and sizing, short tandem repeat typing and profiling, file interpretation and a repeat of the statistical significance of a match if observed [13] . It has been shown [14] that sometime in casework studies or situations such as sexual assault and evidence, DNA mixture may result from a combination of victim and perpetrating body fluids and create a complex and challenging result to interpret.
After PCR amplification, to determine the overall length of the STR, amplicon is sized. This helps to determine the number of repeats present in each allele found in the DNA profile. This is achieved through a sized-based separation involving gel or capillary electrophoresis. Each STR amplification has been fluorescently labeled during PCR, since either the forward or reverse locus-specific primer contains a fluorescent dye; thus by recording the dye colour and migration time of each DNA fragments relative to an internal standard, the size of each STR allele may be determined following its separation from other STR alleles [15] [16] [17] .
There are a number of both biological and instrumental artifacts that often must be sorted out in order to generate a complete accurate STR. As earlier reported [18] [19] known biological artifacts includes vibrant allelic mutation, split peak from incomplete annulation trellis patterns and stutter products [20] [21] [22] . Efforts are ongoing to develop microchips CE platforms to perform high-resolution DNA separations with eventual integration of PCR amplification and CE separation. Moreover, methods such as mass spectrometry (ms) with matrix laser desorption ionization (MALDI) techniques and electrospray ionization (ES) have been used for STR typing without allelic ladder [23] .
Most frequently, thirteen short tandem repeat loci are typically used to create a genetic finger print of an individual in forensic laboratories. While some controversies still exist, the DNA data bases in the United Kingdom uses the National Data base. In the United States, the genetic finger prints are kept at various-combined DNA index systems (CODIS) data bases ranging from the smaller local levels to national levels. They have additional benefits of being able to successfully monitor patients following transplant therapy and it is also used to detect or allow for the prediction of problems associated with transplant, such as graft rejection or relapse of the disease.
In this study, we have used 16 STR loci to optimize the use of the powerplex (R) 16 Hs system to elucidate the DNA profiles in a paternity dispute.
2. Materials and Methods
Whole blood was collected from alleged Fathers X and Y and the Child in dispute, Z after obtaining their consent. A drop each of their blood sample were impregnated on a nucleic acid card (product of
COPAN
) and labeled appropriately with name and laboratory number. The drop of blood was allowed to dry and the blood sport was punched with a 1.2 mm Harris Punch. Sample preparation was done in a Biosafety Cabinet as follows:
Reagent Positive Test Sample(s)
Master mix (µl) 12.5 12.5
Primer mix containing
16 STR (Powerplex TM16) 12.5 12.5
(2000): 1 in 2 × 1017)
1.2 mm blood disk Absent Present
Positive DNA control (µl) 2 Nil
The STR-Short tandem repeats were labeled with specific dye (multiplex STRs with fluorescent dyes). The positive control and test samples (alleged fathers and child) sample was then loaded into the Thermal cycler for polymerase chain reaction (PCR).
The following protocol was adopted in this human DNA amplification using selected short tandem repeat primers labeled with fluorescent dyes. The PCR machine was programmed with the following conditions.
1) Initial denaturation at 95˚C for 11 minutes;
2) Denaturation at 95˚C for 20 minutes;
3) Annealing at 59˚C for 2 minutes;
4) Extension at 72˚C for 1 minute;
5) Go to step 2 for 26 cycles;
6) Final extension at 60˚C for 25 minutes;
7) Final holding at 4˚C forever;
8) 1.0 µl of the amplified DNA fragment(s) was denatured with 14.5 µl Gene scan at 95˚C for 5 minutes. It was then transferred into the auto region of the Genetic analyzer (AB1 PRISM 310) for processing as described by life Technology (France). The gel block region of the analyser pumps the polyacrylamide gel (POP4) into the capillary during electrophoresis. The auto sampler region allows automated loading of the denatured sample onto the separation medium from the sampled plate in a programmed order. The detection region housing the charged coupled device (CCC) camera enhances fluorescence detection.
On subjecting labeled DNA fragment to capillary electrophoresis on polyacrylamide gel support medium (POP4), they migrate at different electrical mobility across the detection window according to their sizes with the smaller fragments moving faster than the large fragment. The specific labeled DNA fragments are excited by the laser as they pass through the detection window to produce fluorescent light of different wavelength depending on the dye [24] . A series of lenses direct and focus the fluorescent light into a spectrograph. The data collection software (Gene Mapper) collects raw data (spectrograph) and converts it into an electropherogram [25] .
3. Result
The result of short tandem repeat analysis loci for the three subjects is involved in this study (the alleged father X, alleged father Y and that of the child Z are compared).
The DNA profiles shown in Table 1 compare the alleged father DNA profile and that of the child. The similarity in their profiles is supported at 15 loci. This result strongly suggests a probability of 99.99% paternity and thus this alleged father is the biological father.
There is a marked deviation in Table 2, in which there is a mismatched at nine loci: D8S1179, D21S11, CSFIPO, D3S158, D16S539, D2S1338, D198433, D5S818 and FGA. Since the probability is 99.9%, the alleged father in this case is
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Table 1. Comparison of the alleged father X and Child Z with STR locus.
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Table 2. Comparison of the alleged Father Y and Child Z loci.
excluded as the biological father of this child.
4. Discussion
We have taken advantage of the efficacy of short tandem repeats in specifically comparing and identifying DNA loci as a tool in DNA analysis.
In this study, we used the powerplex® 16HS system consisting of D8S1179, D21S11, D7S820, CSFIPO, D3S1358, THOI, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, Amelogenin, D5S818 and FGA. STR genotyping was performed by comparison of sample data to allelic ladders and a simultaneous analysis of the 16 STRs and gender identity. Using consensus evaluation method, we observed that the system was accurate, sensitive and above all reproducible. We have used 12.5 µl of primer mixture which is equivalent to (powerplex TM 16 (2000:1 in 2 × 1017). The specificity of the method can be gleamed when are consider the lack of cross-reactivity with a variety of commonly encountered animal species and some microbial pools. This result is made possible through the concurrent use of fluorescent automated sequencers which plays the dual role of the ability to multiplex several loci together. This is again enhanced by the use of polymerase chain reaction which elevates the sensitivity of the method to enable the analysis of cells. As earlier suggested by [26] [27] [28] [29] , technical characteristics of the different fluorochromes groups in multiplexes override the molecular characteristics of each STR in their capacity to detect mixtures. This is further collaborated by the fact as shown by [30] , that sometimes difficulties have arisen in real forensic casework when there are mixed samples which may include extraneous contamination, unknown number of contributors or unbalanced proportion of each one in the sample and a varied degree of degradation of the biological materials contributes extensively in the interpretation of sample profiles. Some earlier works have also confirmed this fact.
Consistent with the potential of STRs to discriminate between inherited regions of our DNA that show variation from person to person, the polymorphic property was taken advantage in this work. The similarity in the seperated patterns in Figure 1 for X and Z elicits marked identity between the disputed child Z and the father X. There is however a sharp contrast between the disputed child Z and the father Y. By examining the STRs, unique evidence emerged to establish relationship in the investigation. As shown in Table 1, the 15 STR showed strong similarity in their profile. This shows the fact that the alleged father is most likely the biological father of the child. A 99.9% similarity is a strong evidence for inclusion.
We observed a marked deviation in Table 2 in which there is a mismatch at the following nine loci: D8S1179, D21S11, CSFIPO, D3S1358, D16S539, D2S1338, D19S433, D5S818 and FGA. This will give a percentage of 56.25 similarity. However, considering the fact that the probability of paternity is 99.9%, there is a strong and compelling evidence for exclusion of this father as a biological father. We show here that despite the fact that humans share overwhelming
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KEY: X―Alleged father; Y―Alleged father; Z―Child.
Figure 1. 1.5% agarose gel electrophoresis image stained with ethidium bromide.
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Figure 2. Spectograph of 16 STRs ran on ABI 310 sequencer with Genemapper ID.
majority of the same DNA, the tiny fraction of a% that varies has been exploited to create a profile of the individual.
In separating the DNA, the procedure used includes PCR amplification and a further determination of the amplicon to have the knowledge of the number of repeat units in each alleles. By using capillary or gel electrophoresis the STRs were fluorescently labeled during the PCR. This was possible since either the forward or reverse locus-specific primer contains a fluorescent dye. To determine the size of each STR allele, we recorded the dye colour and migration time of each DNA fragments in relation to an internal standard. The Applied Biosystem PRISM 310 genetic analyzer and genemapper were used to generate the spectrogram show in Figure 2 elucidating the 16 STRs. Genetic analysis is enhanced using STR matrix as potent markers. The benefits derivable from these techniques have enhanced DNA analysis. In spite of the enormity and varied forms of STRs that are contained in the human genome, only a few set of loci have been selected for use in forensic DNA and human identity testing. It is important to note that core loci allow equivalent genetic information to be shared and compared.
5. Conclusions
The objective of this research was to identify nucleotide tandem repeats in the DNA sequences of the subjects that we have studied. The number of repeats is variable in populations of DNA and within the alleles of individual serving as markers.
The novelty here is a lack of cross reactivity which is commonly encountered in animal studies and makes the expression of polymorphic properties evident.
Acknowledgements
We acknowledge and strongly appreciate the support provided by Lahor Laboratories for the use of equipment and reagents to enhance the study.