The Development and Applications of Short Tandem Loci in DNA Tests - Essay Example

The Development and Applications of Short Tandem Loci in DNA Tests Introduction The evolution of DNA applications in making absolute identifications on human beings has come a long way to develop into advanced stages that can provide adequate information on the identification of seriously dilapidated bodies. Before these breakthroughs, the process of identifying various people involved in various disastrous events such as burning, rot/ decay, etc. has been an uneasy task to many. These complications have brought in a lot of confusion and disputes among various people of the world. With the dawn of the application of DNA tests, the scientific applications have been the most preferred methods of conducting such confusing and technical procedures. Many people across the globe have received fair justice by easily comparing their DNA samples with that of their relatives involved in different disastrous events such as those mentioned. Such mass fatal incidents can lead to various disputes among different people should the process of identification not be handled accurately and with a lot of expertise. Disasters such as fire tragedies involving many people often cause serious body disintegration of those involved in such ordeals. These often cause gross challenges in the process of identification and mars forensic enquiries over the same (Drabek et a.,2004). In situations such as these, the extent of complexity in the process of identifying these concerned bodies lies on the extent of dilapidation caused on the bodies. Besides, in such cases (Graham, 2006), only a few bodies’ fragments are recovered from such happenings making it further difficult for the investigators to try and find out the various persons involved in the ordeal. Moreover, in certain cases, the recovered body fragments tend to be very much fragmented to the extent that their identification using the traditional methods yields no fruit. Under such circumstances, historical methods of identification such as visual identification procedures may not yield much to the success of the entire process. Other identification procedure adopted of late such as finger prints records/ match dental/ the use of X- ray techniques (Hill et al., 2008) among other tend to yield not many fruits under such circumstances resulting into many confusing results hence conflicts (Krenke et al., 2002). The discovery of DNA applications has proved these beneficial to conduct these affairs making the process cheap and reliable as compared to the others. Based on this reason, this paper aims at justifying the reliability on the development and applications of Short Tandem Loci (STR) KITS to generate DNA profile from highly degraded and compromised samples resulting from the mass disaster. I will also discuss the role of STR kits in mass disaster victim identification as well as describing the salient features in the development of the same to its present status. What is Short Tandem Repeats (STR) Loci? Short tandem repeats have been used in forensic investigations and medical qualifications over a wide array of cases where traditional methods of identification cannot be applicable. The method, also referred to as simple sequence repeats (SSRs) or micro- satellites (Lygo, et al., 1994) comprises of sets of accordion- like strands of DNA bearing repeat units consisting of around two and seven nucleotides. These nucleotides as Mulero, et al., (2008) notes, are made up of repeated tandems ranging from approximately half a dozen to several dozens on the same strand. The procedure operates on the basis of the multiplex PCR amplifications whereby the DNA samples bearing strands of variant STR alleles are separated by the method of capillary electrophoresis. These alleles are then compared to the allelic ladder provided from the commercial kit. This aids in accurate identification of the DNA characteristics of the different persons involved in the incidents (Hoff-Olsen & et al., 1999). As Alonso, et al.,(2005) notes, this scientific method make use of just a few sets of STR markers in order to carry out the investigations in a complete manner. This is so despite the fact that the human body contains thousands or even millions of STR markers. These selected STR loci are suitable in providing adequate and relevant information on the DNA characteristics of the individuals being tested making accurate the identification process (Alshamalia, et al., 2004). A lot of commercial kits are available today in the market which can be used in carrying out the entire process. This method is today used by many people, agencies and learning institutions for various purposes such as criminal identification, teaching and instructional purposes as well as to aid in forensic investigations. As a result, several breakthroughs and developments have been made in the process. The history of Multiplex kits Multiplex kits are commercially available kits which are used in DNA testing and applications to conduct DNA tests with the help of short tandem repeat (STRs). The origin and development of these applications according to (Butler, et al., 2003) can be traced to several years in the course of scientific developments. The first multiplex kit is believed to have been developed in Europe by a scientist known as Ray White in 1980 (Butler, 2003). White described the first polymorphic RFLP marker which formed the pioneering work in the development of such kits to be used in STR applications and consequently in DNA testing and personal identification measures. This was followed by a number of other pioneering developments which as well helped in the development of the present day scientific applications in various fields of science. Such include the works of Alec Jeffreys who discovered the multilocus VNTR probes in 1985 (Clayton et al., 1995). This led to the publication of the first paper ever providing details on the development in the same field. Subsequent publications with different measures and procedures detailing the entire process and the suitability of using it in DNA test captured the interests of the FBIs. They Therefore began the DNA case works in their research centers in order to come up with possible aids for forensic investigations. The results generated thereafter prompted the publication of various breakthroughs in the field which aided in the development of the multiplex kits that we have today. During this time, there were many STR multiplex kits developed in forensic genetics. Corach, Risso, Marino, Penacino, & Sala, (2001) observe that the Quadruples were developed for the first time by Forensic Science System by amplifying only 4 loci in the same reaction. This was then exchanged with the SGM kit to amplify 7 loci (Opel, et al., 2006). From 1998, the Amp STR SGM Plus (applied Biosystem) has been used by most of the laboratories to amplify 13 loci which is applied for forensic casework in various parts of the world today (Oslen et al., 1999). Further developments into the matter led to the discovery of the Identifier and PowerPlex 16 kits in the USA which are nowadays in most forensic investigations all over the world (Romano et al., 2006). Today, several companies and research bodies have come up with different types of multiplexes which can be applied in different situations around the globe. Several commercially available kits contain mixtures of polymerase, enzyme buffers and dNTPs (Schwark et al., 2005) for the generation of STR profiles used in the any criminal and contradictory cases (Westen & Sijen, 2009). Table 1 above shows some of the commercially available multiplex kits across the global markets and their salient characteristics distinguishing each from the other. The diagram below shows how DNA cells are arranged in a DNA strand and the regions used for STR analysis using multiplex skits; It is the target region within the DNA strand (Asamura, Sakai, Ota, & Fukushima, 2007) that is often used for extracting the repeat alleles used in the STR tests. The repeat alleles used in such tests are arranged in a DNA strand as shown in the diagram below The most remarkable developments in the history of STRs is associated with the founding of the Quadruplex kit which was the first STR DNA profiling systems to amplify 4 STR loci (HUMVWA31/A, HUMTHO1, HUMF131A1 and HUMFES/FPS) (Aznar et al., 2004). This system was capable of distinguishing a mixture ration of between 1:2 to 2:1 (Budowle et al., 2011). The first DVI projects to engage DNA profiling technology was carried out in 1993 during the Waco siege following a fire break out in1993 (Camacho, Vieira-Silva, Dario, Ribeiro, Espinheira, & Geada, 2008). A DNA profiling was performed on 61 bodies, resulting into a positive identification of 26 bodies while the remaining bodies remained controversial in their identity. This led to the development of the STR DNA profiling which was then used in carrying out the DVI in 1996 following the Spitsbergen aircraft disaster. Short-tandem repeat (STR) technique for 8 loci, of 257 body parts, approved the regeneration of the all 141 dead bodies expressing a 100% success rate for STR DNA of the provided samples (Constantinescu et al., 2012). Following the inventions of Multiplex PCR amplification of (STRs) loci, great success have been achieved with the multiplex currently playing a very important role in mass disaster identification of the victims. This is due to its accuracy and power discriminating fine variations in finer details. The development of more accurate STRs (Donnelly & Friedman, 1999) is concerned more with increasing the number of STR loci used in the test in order to increase their discriminatory power and provide finer details which can be used undisputedly. This trend led to the development of the miniSTRs which have been used to provide even finer details which could not be properly distinguished by the prior methods. The miniSTRs The miniSTR primer sets have been developed into many miniplexes. The initial plexes bore between three to six loci. The miniplex sets have proved useful in obtaining fully amplified loci profiles from degraded and highly compromised samples, where allele dropouts appear with the use of standard commercial STR kits (Fondevila Et al., 2008b). MiniSTRs have strengthened the chance of producing STR results from compromised, inhibitory and degradation samples. It is highly sensitivity for longer loci which would otherwise generate poor profiles with other multiplex kits. The miniSTR profiles obtained include 8 of the regions with amelogenin, the gender determination allele, as standard STR. If no profile or partial results are obtained from standard STR DNA analysis it may be possible to perform the sample for miniSTR kit. The results generated here can then be compared with those from other multiplexes in order to obtain a full DNA profile for the compromised or degraded samples. It is therefore revealed that miniSTR kits can make it possible to obtain profiles for burned and compromised human remains (Camacho, Vieira-Silva, Dario, Ribeiro, Espinheira, & Geada, 2008). Mini STR kits for Human Identification were developed owing to the knowledge of the loci used in STRs. These loci are usually covered by standard short tandem kits showing differentiation in the different alleles. A DNA test conducted using miniSTR for a homicide victim using a resolution of 9 loci demonstrated that MiniSTRs obtained dependable results compared to STR which provided just partial STR DNA profiles which would not be very helpful in making accurate distinctions. Since then miniSTRs became the most robust and sensitive loci to be used for many extremely damaged and degraded samples especially in fire disaster, earthquakes as well as for water based victims (Alshamalia et al., 2004). However, other major breakthroughs have been invented in an attempt to improve the capacity of the miniSTR in making even more clear distinctions. This led to the development of the MiniFilerTM kit with 9 loci compared for better discriminative capacities. Compared to the AmpFSTR_ Identifier_ and SGM Plus, it has been revealed that the MiniFilerTM kit produces the best amplification result of compromised DNA samples. The interpretation rule covered the peak heights, peak height ratios, peak balance and the absence of nonhuman DNA peaks. The use MiniFilerTM kit is capable of detecting even small amounts of non- probative and inhibitions. Based on this fact therefore, it is considered to be greatly helpful for amplification compared to other STR kits. Besides, it can be performed for human remains and mass disaster victims’ identification (Clayton, 1995). The Y STRs The demands for even more accurate results have as well led to the development of the YSTRs. These are based on the test specifically for the Y chromosomes in an attempt to generate clearer discriminations. These markers are only used for male discrimination since the Y chromosome is only found on male beings and not females. These markers are very important in mass disaster identification of missing person as the y chromosomes are passed from father to son without any changes (Constantinescu & et al., 2012). There are two commercial kits of Y STR multiplexes developed; these include the PowerPlex Y (Promega Corporation) with 12 loci and AmpSTR Y filer (Applied Biosystems) with 17 loci in order to increase the discrimination power of the technique (Drabek et al., 2004). Y STRs markers are very valuable and supply additional power that would not be obtained by standard STRs kit alone. This test was used in making discrimination for a fire explosion where burned human remains of nine victims were found, five of the bodies were highly compromised and degraded such that they could not be identified by their relatives. One of the body remains failed to obtain a standard STRS profile. Y STRs were performed and it was possible to match the remains with his brother. Y STRs method can today be used where standard STRs markers fail to do so or to obtain DNA profile from highly compromised samples. However, Y STR is considered a less powerful tool because an individual’s father, paternal grandfather, brothers and sons will all share the same Y chromosome alleles unless the mutation has altered some. The procedure however provides satisfactory assistance in resolving complicated paternity and mass disaster cases (Fondevila Et al., 2008b).The area of Y-STR examination and its purpose to forensic genetic has gone through good development in last few years. Commercially available kits now allow us to easily perform Y-STR profiling, there were more than 200 Y-STRs available. Effects of choosing DNA extraction methods in generating profile compromised samples Various methods have been suggested for use in extracting profiles from highly compromised/ degraded specimens. Five of these have been tested by (Hoff-Olsen & et al., (1999) and their effectiveness in providing reliable results determined. These included; the phenol–chloroform method, silica based method, InstaGene MatrixE (BioTest) method, glass fiber filter method and Chelex based method. The phenol- chloroform and silica based methods proved very costly and time consuming as well as yielding limited results in a range of tests. On the other hand, the chelex method was found to be less expensive, less time consuming as well as yielding greater results while the silica gel method proved very suitable in yielding better results especially when the biological sample tested is of a human being. The choice of any method will therefore be determined by the amount of profiles that can be generated, duration of the procedure and the cost of using the method. Need for the use of better buffers, need for enzymes and new developments for DNA repair Different buffers used in DNA repair have generated different range of results depending on the number of profiles that can be distinguished over s given time. While some buffers e.g. the miniSTRs have shown more reproducible results averaging to about 60% of the alleles detected for a period of 120 minutes, others such as restorase (PreCR) have indicated very poor detective capabilities. However, none of these buffers has been able to utilize the maximum amount of DNA recommended by the manifcaturers with restorase incubation only being performed with just 5ng of DNA out of the 50 ng recommended by the manufacturers. An experiment conducted by Westen & Sijen, (2009) to etermine the effectiveness of these bufers revealed that an increase in the length of exposure to UV radiations only degraded the DNA rather than leading to more alleles being detected. It is therefore recommended that more powerful bufers be developed which can yield more accurate results compared to the ones existing to date. Future developments The future developments are aiming at developing STRs with longer loci for clearer discriminations in order to provide better results beyond the earlier discoveries. An example in the development, in this direction, is the non CODIS miniSTR loci which were are capable of amplifying short alleles of up to (50-150 bps). CODIS loci are currently used as a complement for the degraded and compromised samples as well as parentage testing with the relative reference sample. It is important to develop more miniSTR loci because all the human genome’s chromosomes posse unique repeats and current standard DNA profiling kits own only 14 out of 22 autosomal chromosomes. The purpose of these developments is to produce Rs with wider multiplexes and many loci available in different dye sets to increase the efficiency rate of these loci and obtaining more discrimination in complicated casework such as missing person and mass disaster victim identification. Some of these miniSTR loci are being generated in many laboratories around the world as a tool to produce acceptable profiles from biological evidence that produce incomplete, partial profiles performing standard STR technique (Schwark et al., 2005). Conclusion In conclusion, it is good to mention here that the developments in DNA discrimination techniques have made it possible to achieve relevant and more accurate solutions to various indent problems. The discovery of STRs is one of the most remarkable breakthroughs in science as far as the identification of persons with degraded body parts is concerned. The success of these scientific methods in conducting DNA tests have as well led to further developments to enhance the clarity of the information generated and avoid much disputes. This is marked by the developments in the miniSTRs that use multiplexes to discern differences between various damaged samples as well as making clear distinctions. Further developments into the same sphere have as well led to the generation of the Y STRs used in male discrimination using the Y chromosomes. Further developments in this sector, through research, aims at producing other plexes that would yield better results. References Alonso, A., et al. (2005). Challenges of DNA profiling in mass disaster investigations. Croatian Medical Journal, 46, 540- 548. Alshamalia, F., et al. (2004). Y Chromosome in forensic casework and paternity testing. International Congress Series , 1261, 253-256. Asamura, H., Sakai, H., Ota, M., & Fukushima, H. (2007). "MiniY-STR quadruplex systems with short amplicon lengths for analysis of degraded DNA samples". Forensic Science International: Genetics , 1 (1), 56-61. Aznar, J., et al. (2004). "I-DNASE21 system: Development and SWGDAM validation of a new STR 21-plexus reaction". Forensic Science International: Genetics , 8 (1), 10-19. Budowle, B., et al. (2011). "Population genetic analyses of the NGM STR loci",. International journal of legal medicine,, 125 (1), 101-109. Butler, J. M. (2003). Recent developments in Y-short tandem repeat and Y single nucleotide polymorphism analysis. Forensic Science Review, 15, 91-11. Butler, J. M. et al., (2003). The development of reduced size STR amplicons as tools for analysis of degraded DNA. Journal of Forensic Sciences, 48 (5), 1054-1064. Camacho, S., Vieira-Silva, C., Dario, P., Ribeiro, T., Espinheira, R., & Geada, H. (2008). "Mini-SGM multiplex in degraded samples",. Forensic Science International Genetics Supplement Series, 1 (1), 100-101. Clayton, T. (1995). Further validation of a quadruplex STRDNA typing system: a collaborative effort to identify victims of a mass disaster. (M., Whitaker, J.P., Fisher, D.L., Lee, D.A., Holland, M.M., Weedn, V.W., et al.,et .a.l., Eds.) Forensic Science International, 76. Constantinescu, C., et al. (2012). "Challenging DNA samples solved with MiniSTR analysis. Rom J Leg Med, 20, 51-56. Corach, D., Risso, L. F., Marino, M., Penacino, G., & Sala, A. (2001). Routine Y-STR typing in forensic casework. Forensic Science International, 118, 131-135. Donnelly, P., & Friedman, R. (1999). "DNA database searches and the legal consumption of scientific evidence. Michigan law review, 97 (4), 931-984. Drabek, J. et al. (2004). Concordance study between miniplex STR assays and a commercial STR typing kit. Journal of Forensic Sciences, 49 (4), 859-860. Fondevila, M., Et al. (2008b). "Case report: identification of skeletal remains using short-amplicon marker analysis of severely degraded DNA extracted from a decomposed and charred femur". Forensic Science International: Genetics, 2 (3), 212-218. Graham. E.A.M. (2006). Disaster Victim Identification. Forensic Science Medicine & Pathology , 2, 203-207. Hill, C. R. et al. (2008). Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. Journal of Forensic Sciences, 53, 73-80. Hoff-Olsen et al. (1999). Extraction of DNA from decomposed human tissue: An evaluation of five extraction methods for short tandem repeat typing. Forensic Science International , 105 (3), 171-183. Krenke, B., Tereba, A., Anderson, S., Buel, E., Culhane, S., Finis, C., et al. (2002). Validation of a 16-locus fluorescent multiplex system. Journal of Forensic Sciences , 47, 773-785. Lygo, J. E., Johnson, P. E., Holdaway, D. J., Woodroffe, S., Whitaker, J., Clayton, T. M., et al. (1994). The validation of short tandem repeat (STR) loci for use in forensic casework. International Journal of Legal Medicine , 107, 77-89. Mulero, J. J., et al. (2008). Development and validation of the AmpF/STR Minifiler PCR amplification Kit: A miniSTR multiplex for the analysis of degraded and/ or PCR inhibited DNA. Journal of Forensic Sciences , 53, 838-852. Opel, K. L., et al. (2006). The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains. Journal of Forensic Sciences , 51 (2), 351-356. Oslen, P., et al. (1999). Extraction of DNA from decomposed human tissue: An evaluation of five extraction methods for short tandem repeat typing. Forensic Science International , 105, 171-183. Romano, C., et al. (2006). A novel approach for genotyping of LCN-DNA recovered from highly degraded samples. International Congress Series , 1288 (1), 577-579. Schwark T., et al. (2005). Reliable genetic identification of burnt human remains. Forensic Science International: Genetics , 5 (5), 393-399. Westen, A., & Sijen, T. (2009). Degraded DNA sample analysis using DNA repair enzymes, mini-STRs and (tri-allelic) SNPs, Forensic Science International. Genetics Supplement Series , 2 (1), 505-507. Read More