Evaluation of two MiniSTR loci mutation events in five Father-Mother-Child trios of Yoruba origin

The robustness of short tandem repeats for use in forensic and paternity depends on their high polymorphism and mutation rate. This study tried to determine the event of mutation of two miniSTR loci in the Yoruba population. Blood samples were collected from five father-motherchild trios of Yoruba origin. Two DNA extraction methods, an homemade method and Zymogen gDNA kit were tested for yield and purity for use in the STR assay. The DNA were amplified and resolved on 4% Agarose gel. The first DNA extraction method yielded an average DNA concentration of 1399 ng/μl and while the Kit yielded 984.1 ng/μl; absorbence quotient at 260/280 of 1.78 and 1.55 respectively. Locus D1GATA113 was detected in the father and mother of two families; A and C. D5S2500 was detected only in the male parent (father) in family D. DNA extracted using any of the two methods in this study is appriopriate for use in STR mutation assay but the PCR condition for mutation miniSTR loci among the yoruba still requires extensive


Introduction
some STR in the regulation of transcription of genes like Genetic identity testing involves identifying the epidermal growth factor gene (Gebhardt, et al patterns of genetic material that are unique to 1999), P-53 Inducible Gene-3 (Contente et al., 2002) almost every individual.Short tandem repeats and the regulation of gene expression (Heale and Pete, (STRs), which according to Schoske et al., (2003Schoske et al., ( ) 1995)), have been reported.The mutation rates in STR are repeating DNA sequences which are sequences are several orders of magnitude higher -6 -2 approximately 2-6 base pairs in length; are the ranging from 10 -10 nucleotides per generation in most commonly used loci for human identification human (Fan and Chu, 2007), than in 'regular' DNA simply because of their high polymorphism, sequences in the genome (Ellengren, 2000).The requirement of minimal template DNA and chances of detecting germline genomic mutations have possession of a narrow size range that makes also increased with the use of STR markers due to their multiplexing easy (Butler, 2005;Opel et al., 2006).
higher mutation rate when compared to other genetic Most STRs are found in the noncoding markers.STR systems are nowadays the most regions, while only about 8% locate in the coding commonly used systems in human identity and regions (Ellengren, 2000).About 90% of STR paternity cases.from samples with compromised DNA.This involves microlitres of each blood sample was pipetted into 1.5 the redesigning of the primer binding sites of some ml eppendorf tube and then 1000 µl of red cell lysis STR loci to produce shorter amplicons (Gill et al., buffer was added.The tubes were spun for 2 minutes at 2006).The miniSTR primer sets produce full genetic 7000 rpm and supernatants discarded The steps were profiles in the majority of the samples containing repeated until all hemoglobins were removed.The degraded DNA (Butler, 2005); and have the tubes were allowed to drain after which 400 µl of nucleic potential to provide additional discrimination in lysis buffer was added to the eppendorf tubes and complex paternity cases or missing persons cases vortexed till all pellets were dissolved, followed by the (Goodwin et al., 2004).
addition of 100 ul of saturated NaCl (5M) and 600 µl of There has been no data on miniSTR chloroform and spun for 2 minutes at 7000 rpm.400 µl analysis on Nigerian populations most especially of supernatant was transferred to a new 1.5 ml tube and the Yorubas.This study tends to fill literature gap 800 µl of cold (-20°C) absolute ethanol was added to and provide foundational data on miniSTR analysis precipitate DNA and vortexed.The tubes were spun for in Yoruba population of Nigeria, by evaluating the one minute at 12000 rpm, and then the supernatant presence of two miniSTR loci and the event of discarded carefully.Fifty microlitres of TE buffer was mutation on the Yoruba population.
added and then vortexed.The eppendorf tube with DNA was kept at 4°C.

Materials and Method
For the second method using Genomic DNA The PCR was carried out using the Gene Amp® PCR   many samples recovered from crime scenes yield only few nanogram or picogram amounts of DNA that is The few amplicons produced also couldn't separate sometimes degraded (Edwards, et al., 1992).Based on very well for us to see the alleles of the band and amount of DNA, both methods are still proficient for use this improper separation is the same of the ladder in STR mutation assays.However, purity of DNA is of marker that was used in the electrophoresis (fig 4).
greater concern as contamination of samples (Lorenz, 2012) by RNA, protein(s), enzymes like nuclease(s) may yield poor amplification.Absorbance quotient value ranging from 1.8 -2.0?, is indicative of good and purified DNA (Ghatak et al. 2013).This implies that Iranpur and Esmailizadeh) extraction method yielded the purest DNA of the two methods.Though, on the overall, the amplicon frequency was low in both methods, which probably means the methods have nothing to do it the frequency of amplicons recorded.Another observation of interest that featured in our result is the presence of smear in fragmentation of DNA should not influence miniSTR mutation results which have reduced-size amplicons Discussion (Butler, 2005).

DNA fingerprinting has been very helpful in
The allele sizes of the amplicons are compared forensic identification especially the use of miniSTR.
between parents and off-springs to determine if However, the marker to use must first be validated mutation occurred.In this study, loci D1GATA113 could for its polymorphism within the population.The only be detected in two members of family A and C, and DNA concentration average value of 1399 ng/ml loci D5S2500 detected only in the father of family D. The and 984.1ng/ml obtained from the two extraction lack of amplification in all the families may be as a result methods for this study cannot be said to be too low of inappropriate PCR conditions like reagent concentrations, for STR mutation analysis.Though, the amount of cycling conditions (Lorenz, 2012).Commonly implicated DNA obtained using the Iranpur and Esmailizadeh's reagents whenever a standard PCR conditions did not (2014) method is higher than the yields from the yield the desired amplicons are the Mg2+, primer extraction kit, which may be as a result of the concentration or both.The contamination of DNA or difference in the amount of blood used to get a 50 ul reagents used could also result in low amplicon yield. of dissolved DNA.The Iranpur and Esmailizadeh, The PCR products, could not separate well in 4%

Fig 1 :
Fig 1: A Gel representing DNA concentration of samples extracted using Kit in agarose gel electrophoresis.
Fig 2: A Gel representing DNA concentration of samples extracted using the homemade method in agarose gel electrophoresis.The alphabets from the labellings represent the Family tags while the numbers represent members of the family father, mother, child respectively.The result of the PCR amplification after running in 4% agarose detected loci D1GATA113 method requires 500 ul of blood, while the extraction kit (Fig 3) in two families though not up to a trio set in used up 100 ul of blood in each sample to give 5ul of each family and loci D5S2500 was detected in one DNA in solution.STR was specifically designed because family (Fig 4).manysamples recovered from crime scenes yield only few nanogram or picogram amounts of DNA that is The few amplicons produced also couldn't separate sometimes degraded(Edwards, et al., 1992).Based on very well for us to see the alleles of the band and amount of DNA, both methods are still proficient for use this improper separation is the same of the ladder in STR mutation assays.However, purity of DNA is of marker that was used in the electrophoresis (fig4).greaterconcern as contamination of samples (Lorenz, 2012) by RNA, protein(s), enzymes like nuclease(s) may yield poor amplification.Absorbance quotient value ranging from 1.8 -2.0?, is indicative of good and purified DNA(Ghatak et al. 2013).This implies that Iranpur and Esmailizadeh) extraction method yielded the purest DNA of the two methods.Though, on the overall, the amplicon frequency was low in both methods, which probably means the methods have nothing to do it the frequency of amplicons recorded.Another observation of interest that featured in our result is the presence of smear in Fig 4: Amplification D5S2500 in family C.This samples extracted with the kit, which is an indication of ladder is showed the amplicon of primer D5S2500 fragmented DNA samples and when in comparison with on Family D-father alone and nothing on the mother Iranpur and Esmailizadeh method was inferior.But the (DM) and Child (DC) of the same family.fragmentation of DNA should not influence miniSTR mutation results which have reduced-size amplicons Discussion(Butler, 2005).DNA fingerprinting has been very helpful inThe allele sizes of the amplicons are compared forensic identification especially the use of miniSTR.betweenparents and off-springs to determine if However, the marker to use must first be validated mutation occurred.In this study, loci D1GATA113 could for its polymorphism within the population.The only be detected in two members of family A and C, and DNA concentration average value of 1399 ng/ml loci D5S2500 detected only in the father of family D. The and 984.1ng/ml obtained from the two extraction lack of amplification in all the families may be as a result methods for this study cannot be said to be too low of inappropriate PCR conditions like reagent concentrations, for STR mutation analysis.Though, the amount of cycling conditions (Lorenz, 2012).Commonly implicated DNA obtained using the Iranpur and Esmailizadeh's reagents whenever a standard PCR conditions did not (2014) method is higher than the yields from the yield the desired amplicons are the Mg2+, primer extraction kit, which may be as a result of the concentration or both.The contamination of DNA or difference in the amount of blood used to get a 50 ul reagents used could also result in low amplicon yield. of dissolved DNA.The Iranpur and Esmailizadeh,The PCR products, could not separate well in 4%

Table 2 :
DNA concentration summary in ng/ul.

Awe et. al. / Nig. J. Biotech. 33 (2017) 120-124
Fig 3: Amplification of D1GATA113 in family A. This figure showed amplicons on AF (Family A-father) and AM (Family A-mother) but nothing showed on Family A child (AC).agarosegel.The improper separation of the 20 bp extraction from Human samples for PCR-RFLP analysis.ladder used as rule marker confirmed this.This J. Biomol.Tech., 24:224-231 result contradicts White and Kuskawa, (1997) reports that agarose gel is sufficient for analysis of Gill, P., Fereday, L., Morling, N., and Schneider, P. (2006).samples for several commonly used STR loci.This The evolution of DNA databases recommendations for probably can also be because, the miniSTR are new European loci.Forensic Sci.Int.156: 242-244.Hill, et al. 2008; Iranpur and International Congress Series, 1261: 460-2.Esmailizadeh, 2014).Since the alleles didn't come up probably because inappropriate PCR conditions Heale, S.M. and Petes, T.D. (1995).The stabilization of and improper separation of the agarose gel used, repetitive tracts of DNA by variant repeats requires a the allele bands could not be scored.functional DNA mismatch repair system.Cell 83: 539-This study reveals that DNA extracted 545.using any of the two methods in this study is appriopriate for use in STR mutation assay.Hill, C. R., Kline, M. C., Coble, M. D., and Butler, J. M.However, the PCR condition for detecting the type (2008).Characterization of 26 MiniSTR Loci for and frequency of mutations of miniSTR loci among Improved Analysis of Degraded DNA Samples.J. the Yoruba population still requires extensive Forensic Sci., 53(1): 1556-4029 optimization.Most importantly, this is the first study to detect miniSTR loci in a Yoruba population.Iranpur M. L., and Esmailizadeh A. K. (2014).Rapid Extraction of High Quality DNA from Whole Blood Stored