Over the past few decades, there has been a growing interest in applying DNA profiling tools in forensic analysis. One of such tools include single nucleotide polymorphism (SNP) profiling which can be used for defining mitochondrial DNA (mtDNA) and Y chromosome as well as for checking autosomal SNPs. Specific interest has been drawn from the powerful application of the technology in autosomal SNPs where the tools are used in confirming paternity. The technique takes advantage of the low mutations and degraded sample analysis by the use of short amplicons (Sobrino, Brion & Carracedo, 2004).
As more efficiency, accuracy and reduced processing time become important consideration in techniques used in forensic analysis, newer methods of SNP genotyping are continuously being researched on. Since these technologies have produced numerous techniques for SNP profiling and other DNA typing methods, it almost becomes impossible to select the most effective technique to use in forensic analysis considering the available options. This paper will provide a critical evaluation of SNP as a method of DNA profiling paying specific importance in identifying the advantages and disadvantages of SNPs.
DNA Profiling: A Critical Evaluation of the Forensic Applications of Single Nucleotide Polymorphisms (SNPs) Introduction DNA profiling also known as DNA typing is a technique commonly used in molecular genetics analysis to identify different patterns of DNA. DNA (Deoxyribonucleic acid) is the genetic material that contains the blue print of an individual’s life. DNA is found in all cells and it carries chemical and biological information that is passed from parents to offspring. The human genome (the total genetic information in an organism) is almost identical in all ethnic populations.
However, individuals differ from one another by small differences in their DNA patterns. The ability of DNA profiling or DNA typing of identifying differences in DNA patterns has gained significant applications in modern genetic forensic analysis. The technique can be used to identify criminals and provide evidence for judicial purposes. Although the technique has numerous applications in identifying various types of criminals, DNA profiling has widely been used to identify rape and murder criminals.
The technique has been utilized to find answers to convict a criminal after many years of failed proof of evidence. DNA profiling has also gained many applications in paternity tests where family relationships are determined in the forensic laboratory. Immigration disputes have now become easy to solve than never before (Sobrino, Brion & Carracedo, 2004). The application of DNA profiling in forensic investigations takes advantage of specific locations in the human DNA known as introns. Introns do not code for any proteins and their function is poorly understood in living organisms.
The non coding regions involve several repetitive sequences of DNA which are polymorphic in nature (they have a varying number of repetitive lengths. These varying lengths of DNA sequences have been utilized by a technique known as restriction fragment length polymorphism (RFLP). The information obtained from analysis of non-protein coding DNA is solely used for forensic analysis and may not be used for testing presence of genetic diseases. Disease conditions in man are caused by alteration in protein structure and functions. Proteins are coded by protein-coding parts of DNA known as exons.
Therefore, the analysis of introns will not reveal pathologies but will only help in forensic investigations (Sobrino, Brion & Carracedo, 2004). Over the last couple of decades, molecular geneticists have developed the techniques of DNA profiling to have ultimate sensitivity. It is now possible to obtain enormous amount of DNA from a small amount of saliva that is left on a cigarette end. This DNA can then be processed to obtain a complete profile of an individual which is critical for forensic analysis. Before the technology was advanced, scientists required significant amount samples from criminals which were almost difficult to find.
Apart from the small size of sample required in the modern technology, scientists can now process DNA profiles within a very short time. Techniques such as polymerase chain reaction (PCR) which amplifies large amounts of specific short sequences from the human DNA have increased the speed of DNA profiling. Other important techniques that have made possible the fast processing of DNA patterns are tools for DNA fingerprinting (Sobrino, Brion & Carracedo, 2004). All in all, these techniques have both improved on the sample size as well as the required quality of DNA for analysis.
DNA Profiling: Overview of Methods The technique of DNA profiling utilizes a number of typing systems which include restriction fragment length polymorphism (RFLP) profiling, mitochondrial DNA (mtDNA) analysis, gender profiling, human leukocyte antigen (HLA) profiling, short tandem repeats (STR) profiling, Y-chromosome profiling and single nucleotide polymorphism (SNP) profiling. Early approaches of DNA profiling utilized varying numbers of tandem repeats (VNTRs). VNTRs consist of the repeating sections of DNA sequence which in most cases vary from one individual to another (Sims & Ballantyne, 2006).
The varying DNA sequence units are analyzed as RFLPs which are differences within the specific genomic regions that are specially detected by biological materials known as restriction enzymes. Restriction enzymes detect and cut specific DNA sequences (restriction fragments) according to recognition sites of DNA sequence. The technique RFLP can therefore be said to have been invented after the discovery of restriction enzymes in 1970s. VNTRs are usually 20 to 50 base pairs long per each repeat and an individual can have from 50 repeats to a number of repeats.
Repeats are always inherited meaning they are passed from the parent to offspring and can be used in the forensic analysis. Alec Jeffrey, a British geneticist was the man behind this discovery in 1985 and it now forms the basis of all DNA profiling systems today (Sims & Ballantyne, 2006). Short tandem repeats (STR) are quickly replacing VNTR profiling. The STR regions consist of from 2 to 4 base pair repeats which are usually repeated to about 5 to 15 times. The STR analysis is the modern standard method commonly used in forensic DNA typing.
The advantage of STR is that they have shorter sequence repeats which are much easier to take detailed analysis. All these techniques for DNA profiling utilize the criterion that each human organ, tissue and cell contains similar pattern of DNA and it is easy to develop database that stores information that can be used to identify unique features of every human. The law enforcement and the armed forces in the United States have developed such database that is critical in identifying individuals in a unique manner (Sims & Ballantyne, 2006). Computer technology can allow storage of such critical data and retrieval of the same at all times.
However, the ethical dilemma stands. Before the methods of DNA profiling, there used to be procedures used in identifying humans and animals such as dog tags, dental records and blood typing. Nowadays, modern techniques of DNA profiling have made a major revolution in the field of forensic identification. The newer methods are more conclusive and provide sufficient information about the subject in question –the criminal. For instance, it may be confusing to use blood typing in identifying criminals since there could be a number of individuals with similar blood types.
Dental records may fail in case the dentition is compromised in terms of integrity. It may be damaged to a point of having difficulties in identifying individuals. In DNA typing, DNA can be obtained even when the deceased individual was greatly disfigured. DNA has even been obtained in badly charred bodies that have lost their total integrity. Human hair and bones can also provide critical DNA sample for DNA profiling. This makes the method of DNA profiling to be the method of choice in forensic analysis where available samples are always small and detailed information is required(Pakstis, et al, 2007).
Although the methods of DNA profiling have shown potential improvements compared to the previously used methods in forensic analysis, DNA profiling techniques often encounter some possibilities of making slight mistakes in extracting samples, preparing the samples or analyzing the samples. However, when careful precaution is taken to avoid most of the human errors, the procedure still remains to be the most competent in forensic analysis. It is required that each forensic laboratory maintain an exceptional high quality assurance and quality control standards.
This will prevent common errors in sample extraction, sample preparation and unnecessary formation of artifacts which will compromise the quality of the results. In some states and local forensic institutions, rigors methods are being tried out and standard operating procedures developed to unify the steps involved in criminal identification. Licensing can be made to offering institutions to ensure that only quality institutions can be allowed to offer forensic services to the citizens. This will reduce quarks from practicing forensic analysis (Butler, 2004).
Single Nucleotide Polymorphisms (SNPs) Single nucleotide polymorphisms (SNPs) consist of the most common type of human polymorphisms that exist in the genome (Sobrino, Brion & Carracedo, 2004). It is difficult to state a rough estimate of how many SNPs are present in the human genome. However, in various public and private institutional databases, there are over 5 million SNPs which have been collected in total. Of the 5 million collected SNPs, 4 million SNPs have been confirmed to have great polymorphism in one population or a variety of specific dominant groups of populations.
Perhaps what make SNPs to draw much interest in the area of medicine is their abundance and the simplicity. The nature of SNPs to be limited polymorphic material has particularly attracted significant research into the field of forensic application of SNPs (Sobrino, Brion & Carracedo, 2004). SNPs in medicine have been used in the identification of diseases that are mainly caused by defective genes. SNPs are nowadays utilized in the most amazing and novel study of pharmacogenomics which is the area of genetics involved in testing different response to drug molecules from one individual to another.
It is expected that this science will one time lead to ‘designer’ drugs specifically meant to a particular individual according to his or her genetic constitution. Sobrino, Brion and Carracedo (2004) argue that the development of a haplotype map that has made possible to make a clear definition of variability of the human SNP has been as a result of the completion of the project of mapping the entire human genome. The haplotype map will allow scientists and researchers find genetic variations that result to a number of genetic diseases.
The development of haplotype map will be significant in reducing the number of required SNPs to take a full examination of the whole genome associated with a specific phenotype from 10 million SNP which are assumed to exist to about 500,000 tag SNPs (Sobrino, Brion & Carracedo, 2004). Increased applications of SNPs in forensic investigations are being developed. SNPs have a lot of features that make them suitable for forensic analysis. The most important feature of SNPs is that they have the lowest mutation rates and this feature is crucial for testing paternity.
Another unique feature that makes SNPs suitable for forensic analysis application is that they are suitable for taking analysis by employing high throughput and novel technologies. Despite the potential benefits of SNPs, there are also some specific drawbacks in the using of the technique. The first limitation is that the number of SNP needed is about 4 times as much as the number of STRs that may be required on average. This means that about 60 properly balanced SNPs are required to have the same discrimination power more than the novel complexes that may be in use in the area of forensic science (Sobrino, Brion & Carracedo, 2004).
Another limitation of SNP is the cost benefits which may not still be very clear. These costs especially for high-throughput may be slightly being better than the costs of any commercial kits. However, if one was to compare the cost of paternity test using methods such as STRs, it may be cheaper to use STRs than go for SNPs since STRs are cheaper. It may also be argued that the use of SNP requires new training and careful attention to every detail which might take individuals extra time to learn how to use products.
However, using the STRs methods may become more efficient in these scenarios. Experience of STRs usage among most individuals is almost 10 years. This means that individuals are more conversant with STRs more than they are conversant in SNPs making even the results to be compromised in SNPs than STRs. In a practical scenario when using STRs techniques, mutations in the flanking regions or various polymorphisms have increasingly become known for STRs than for SNPs which might be seen as a bit new technique.
There may be required an extensive population groups validation in the case of SNPs for one to have increased markers (Butler, 2007). It however appears to be a matter of speculation to have SNPs techniques to fully replace the primary method of forensic DNA profiling which is the STRs. However, it may not be denied that there are other powerful applications in SNPs that are not easy to be found in STRs, for instance, the definition of mtDNA haplogroups and the Y chromosomes haplogoups for population group analysis may not be offered by procedures involving STRs.
SNPs provide for the standardizations as well as inter-laboratory assay validations which is prime in the application of SNPs profiling in the area of forensic analysis (Butler, 2007). It may however be conceived that in forensic analysis, there may be no one single method that can be used for SNP typing and for one to choose the most appropriate technique, there should be some factors that may be considered especially those ones emanating from the user of the procedure. These characteristics that are user-defined may include proper multiplexing properties and exceptional accuracy.
In large scale application of SNPs it is also essential to compare the cost of carrying out forensic procedures where the lower the cost of performing DNA profiling, the better the method. Individuals and big forensic laboratories may therefore tens to pick on some methods that may prove to be cheaper and cost effective (Gusmao, et al, 2006). In the marketplace, there are a number of SNPs methods that have been developed to meet some of the requirements that individuals and large forensic companies have ever yearned to have.
Nowadays, it may even become totally difficult to have a choice on the method of platforms or chemistries to employ in carrying out forensic analysis. It is therefore necessary for the users of the technology to have an overview of the technology of SNPs and learn some of the important factors that make SNPs better than other techniques. It is also important to understand the technique of SNPs in details so as to identify some of the weaknesses of applying the technique in DNA profiling Gusmao, et al, 2006). Developments in SNP techniques over the recent decades have seen different improvements in the method of DNA profiling.
These methods that have been developed take advantage of various different types of allelic detections and allelic discriminations platforms. The rapid developments and continued discovery of more advanced techniques have made it impossible for one to critically analyze and choose the most convenient method to use in DNA typing. In order to have a clear understanding on the methods and make a good choice, it is important to understand the differences between various reactions of allelic discrimination. It is also necessary to compare the detection methods and the assay formats.
Usually, reactions of allelic discrimination can be detected with over one type of critical methods. Similarly, same method used to detect materials can be used in the analysis of the products that are obtained with different assay formats or different methods (Gusmao, et al, 2006). Most of the SNP genotyping can however be assigned strictly to one of the four basic techniques depending on the type of molecular mechanism involved (Butler, 2004). These four groups include primer extension, specific allele hybridization, invasive cleavage and oligonucleotide ligation.
Additionally, there are various ways of detecting analyzing different products critical for the process of SNP genotyping; the luminescence, fluorescence, mass measurement and many more. Two categories have been related with the assay formats which are in general referred with the kind of detection used. These are homogenous reactions especially when these reactions take place in solution and the reactions that take place on solid support like on glass slides, beads, chips and much more.
Generally, homogenous reactions have been proved to be more amenable to other automated procedures since there are no purification or separation steps following reactions for allele discrimination. The main drawback in homogenous reactions is that there are limited multiplex potentials. Contrary, the reactions on the solid supports tend to have a greater multiplex potential although further manipulations are needed (Butler, 2007). The Current status and Future Approaches The SNP genotyping techniques have generally been developed rapidly over the past decades.
This has resulted to a variety of different types of genotyping protocols specifically based on SNP. These procedures are nowadays available to researchers and scientists working in forensic and medical laboratories. However, different aspects need to be considered in order to determine which kind of technology is most effective for the purpose of forensic investigation. Forensic investigation requires detained evidence that can provide solutions to be used in the jurisdiction process.
Justice is an important aspect when considering any form of incarceration. Before such detentions are done, it is necessary to ensure that accurate results are obtained to avoid wrong incarceration. The results for forensic analysis should be sensitive, reproducible and accurate. The technology used in DNA profiling has to be flexible, time effective and cost effective (Must, et al, 2008). One of the most limiting factors in all forensic genetic technologies lies in the amount of required DNA for each genotyping.
Some techniques have been developed which interrogate the SNP onto the genomic DNA directly. Pervious techniques used to apply PCR before the real genotyping can be done. The use of PCR is to make complementary DNA (cDNA) which can be used to make various genomic sequences of DNA. Such SNP methods that required PCR included the SNPlex, Invader assay and Illumina. For these technologies that need PCR before any allelic discrimination can be done, it is essential for developing multiplex PCR. This allows analysis of small amounts of DNA with accurate results.
The procedure is also throughput and it provides sufficient information even when the amount of DNA available is small (Must, et al, 2008). The current SNP technologies being used include the minisequencing. These techniques are common in forensic laboratories and allow detection to be done on an automatic instrument with capillary electrophoresis. Capillary electrophoresis is also utilized in STRs analysis. For future developments, new technologies which can be able to reach the expected forensic requirements and criteria need to be developed.
It is often difficult to find one single technology being effective and fully satisfying. Therefore, in future developments, researchers may have to combine the already existing technologies to come up with a novel hybrid technology that may answer some of the unanswered questions in genetic forensic analysis. References: Butler, J. M (2004). SNPs and strips: approaches to rapid screening of mtDNA type. mtDNA workshop: Forensic Human Mitochondrial DNA Analysis. Retrieved May 21, 2010 from: http://www. cstl. nist.
gov/biotech/strbase/pub_pres/ButlerAAFS2004mt. PDF Butler, J. M (2007). Applications of new technologies in forensic genetics. Krakow Forensic Seminar. Retrieved May 21, 2010 from: http://www. cstl. nist. gov/biotech/strbase/pub_pres/KrakowForensicSeminarApr2007. pdf Gusmao, L. , et al. (2006). DNA Commission of the International Society of Forensic Genetics (ISFG): An update of the recommendations on the use of Y-STRs in forensic analysis. Forensic Science International. 157: 187-197. Retrieved May 21, 2010 from: http://www.
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Mutations in brief: Sub-Populations Within the Major European and African Derived Haplogroups R1b3 and E3a Are Differentiated by Previously Phylogenetically Undefined Y-SNPs Retrieved May 21, 2010 from: http://www3. interscience. wiley. com/homepages/38515/pdf/940. pdf Sobrino, B, Brion, M & Carracedo, A. (2004). SNPs in forensic genetics: a review on SNP typing methodologies. Forensic Science International. 154(2): 181-194. Retrieved May 20th 2010 from http://www. bio. unc. edu/courses/2008Fall/Biol423L/ForensicsSci05SNPReview. pdf