Single nucleotide polymorphisms

19 Apr

Single nucleotide polymorphisms

One of the most significant outcomes of the Human Genome Project has been the iden- tification of large numbers of single nucleotide polymorphisms (SNPs) [1-3].

The ap-plication of SNPs to forensic analysis is currently limited to some specialist cases.How- ever, with advances both in our knowledge of SNPs and in the technology used to detect the polymorphisms, SNP analysis may play an increasingly important role in the future.

SNPs – occurrence and structure
‘SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in normal individuals in some population(s), wherein the least frequent allele has an abundance of 1% or greater’ [4]. The structure of a SNP is very simple, an example is shown in Figure 12.1. SNPs are found in the human genome about once in every 1000 bp [1-3, 5]. Given that the human genome is 3.2 billion bp long, we can estimate that there will be approximately 1 million differences between two genomes that are due to SNPs: this represents approximately 85% of human genetic variation. The biallelic state of the vast majority of polymorphisms intrinsically limits the information that can be gained from the analysis of any given SNP, and this has been the major factor limiting their application to forensic analysis: between 50 and 80 SNPs are required to achieve the same levels of discrimination as the current STR based methods [6, 7]. However, the vast numbers of SNPs within the genome (currently over 10 million SNPs have been placed in public databases [1, 2]) can compensate for the limited information carried by any individual SNP, and make them a tempting polymorphism to exploit. The technology that is being used for SNP detection is evolving and SNP analysis is becoming possible in many forensic laboratories.

Detection of SNPs
There are many techniques available for the resolution of SNPs. In the 1970s it was established that particular enzymes produced by bacteria can be used to cut the DNA

Figure 12.1 SNPs are created when the DNA replication enzymes make a mistake as they copy the cell’s DNA during meiosis. The enzyme incorporates the wrong nucleotide approximately once every 9 to 10 million bases. In the vast majority of cases, SNP are biallelic and only have two different alleles. In the alleles shown above, the thymine nucleotide has been replaced by a cytosine molecule by recognizing specific sequences [8]. Restriction digestion can be used to genotype SNPs when the SNP either creates or destroys a particular restriction enzyme recognition sequence [9, 10] but the method is limited for forensic casework because it needs a large amount of DNA and is a long and laborious process.

Sanger sequencing

Sanger sequencing, also known as chain-termination sequencing, was developed in the late 1970s and is a milestone in the development of molecular biology [11]. The sequencing takes advantage of the biochemistry of DNA replication. The first stage of the analysis is to amplify the target region using PCR; amplified products are then used asthetemplateinasequencingreaction.TheDNAsequencingreactionissimilartoPCR amplification and the reaction mixture is very similar, containing the thermophilic Taq DNA polymerase and deoxynucleotide triphosphates (dNTPs). It differs from PCR in that only one primer is used and, in addition to the dNTPs, there are four fluorescently labelled dideoxyribonucleotides (ddNTPs); each ddNTP is labelled with a different coloured dye [12]. The ddNTPs do not contain the hydroxyl group on the 3′ carbon, which prevents any extension of the DNA molecule [13] (Figure 12.2). The concentration of dNTPs is higher than ddNTPs and therefore in most cases a dNTP is added. The ddNTPs are incorporated at random intervals along the molecule. This produces a range of different sized molecules. The products of the sequencing reaction are analysed using capillary gel electrophoresis systems, such as the ABI PRISM® 310 Genetic Analyzer, that separates DNA to single base pair resolution and can simultaneously detect four different fluorescent labels (Figure 12.3). Sequencing is not a practical option for the analysis of SNPs in a forensic context. Most SNPs are widely dispersed around the genome and a separate reaction is required for each SNP. An exception is the mitochondrial genome, where a number of SNPs are concentrated into a small area and can be analysed in a small number of reactions (see Chapter 13). Sequencing has also been a powerful method to type SNPs within rapidly evolving regions of DNA in the HIV virus [14, 15].


Figure 12.2 A primer anneals to the template strand. This is extended by Taq polymerase until a ddNTP is incorporated. The ddNTPs are incorporated at random, which leads to a collection of extension molecules that differ from each other by one nucleotide (shown above labelled 1 to 8). The four ddNTPs are labelled with different fluorescent dyes that are detected during capillary electrophoresis (see plate section for full-colour version of this figure)

SNP detection for forensic applications
Restriction digestion and sequence analysis are not viable methods to use for most forensic cases that might require the analysis of 50 to 80 SNPs dispersed around the genome. A number of methods have evolved that can be applied to the detection of

Figure 12.3 The sequence of a region of the mitochondrial genome. The sequencing software in-terprets the sequence data and ‘calls’ the bases. This information is provided above the sequencing peaks (see plate section for full-colour version of this figure)

multiple SNPs. Methods that are based around the concepts of either primer extension or primer hybridization are the most widely used.

Primer extension
Primer extension is a robust method for discriminating between different alleles and several methodologies have been developed [16]. One of the commonly used methods is the mini-sequencing reaction [17]. The basis of the reaction is very similar to Sanger sequencing. The first part of the procedure is to amplify the target region using PCR. An internal primer then anneals to the denatured PCR product; the 3′ end of the primer is adjacent to the polymorphic site. The primer is then extended by Taq polymerase but only ddNTPs that are labelled with fluorescent dyes are provided; the primer is only extended by one nucleotide. The extended primer can be analysed using capillary gel electrophoresis and the colour of the detected peak allows the SNP to be characterized (Figure 12.4). A widely used commercial kit called SNaPshotTM (Applied Biosystems) is based on this methodology. By using different sized primers and different fluorescent tags for each of the four bases, a large number of SNPs can be simultaneously detected [18].

Figure 12.4 The primer extension assay. (a) The target sequence is amplified using PCR and the products are used as the template in the extension assay; (b) an internal primer hybridizes to the target adjacent to the SNP and a single fluorescently labelled ddNTP is added by Taq polymerase; (c) the reaction is analysed by capillary electrophoresis (see plate section for full-colour version of this figure)

Figure 12.5 Allele specific hybridization. Allele specific oligonucleotide (ASO) probes that include the SNP are hybridized with the target DNA. (a) Under highly stringent conditions only perfectly matched sequences will form stable interactions; (b) with one mismatch in sequence the ASO will not hybridize

Variations on the primer extension technique include pyrosequencing [19, 20]; mi-croarrays, where the extension primers are attached to a silicon chip [21, 22]; and allele specific extension, when the primer is only extended if it is 100% complementary to the target sequence [23].

Allele specific hybridization
Under stringent conditions, even one nucleotide mismatching between a template and primer can differentiate between two alleles (Figure 12.5). There is a large number of methods that exploit the hybridization of probes, including reverse dot blots [24]; Taqman® assays [25]; LightCycler® assays [26]; molecular beacons [27, 28]; and GeneChips® [29] (see Further Reading for details).

Forensic applications of SNPs
A vast amount of data is available on the different SNPs in the human genome and one of the biggest tasks when applying SNPs to forensic applications is to select the most appropriate SNPs from the overwhelming numbers that are available. The choice of SNPs is very much dependent on the application.

Forensic identification
The vast majority of forensic DNA analysis involves the characterization of biological material recovered from the scene of a crime. Several panels of SNPs have been devel- oped that are designed to provide maximum discrimination powers for forensic identi- fication [18, 30, 31]. These contain SNPs that are polymorphic in all major population groups. A panel, containing 52 SNPs was developed by the SNPforID Consortium [18]. Using this panel of SNPs produced match probabilities that ranged from 5.0×10−19 in an Asian population to 5.0×10−21 in a European population. When applied to paternity testing, average paternity indices of between 336000 in Asian populations and 550000 in European populations, were achieved.

However, even with the high discrimination power, the effort involved in analysing 50 SNPs is greater than when undertaking standard STR analysis. The major attraction of using SNPs with the current technology is that SNP analysis can provide results from highly degraded DNA when conventional STR profiling has failed [30, 32].

Prediction of the geographical ancestry

In many cases, the identification of the population group from which a crime scene sample has come from can be valuable intelligence for the investigating agencies: was the person who left the material at the crime scene of Caucasian, Asian, African, mixed ancestry? Panels consisting of mtDNA SNPs and Y SNPs have already been found useful for this purpose [33, 34] but are intrinsically limited by the fact that they can only provide information on either the maternal or paternal ancestry. Autosomal SNPs thathavedifferentfrequenciesindifferentmajorpopulationgroupscanprovidevaluable informationongeographicancestry[35].ManyoftheSNPsselectedforthispurposeare associated with coding regions that have been subjected to selection pressures. These include pigmentation genes and genes involved with the metabolism of xenobiotics. The pigmentation genes, in addition to providing information on geographic ancestry, can also give information on phenotype of the person who deposited the biological material at a crime scene, including skin, hair and eye colour [36-38].

SNPs compared to STR loci
Current STR-based multiplex kits like AMPFISTR Identifiler® and PowerPlex® can amplify 15 STR loci and the amelogenin locus. Using the current technology it is difficult to co-amplify and detect any more STR loci. Also the size of the amplicon for each STR is quite large. The great advantage STRs have over SNPs is their power of discrimination due to the large number of alleles they have in comparison with biallelic SNPs. In contrast to STRs, around four-times more SNPs are required to reach the discrimination power equivalent to STR loci. Another major disadvantage to using SNPs is that mixtures of two or more people might be either problematic or impossible to interpret since SNPs are biallelic markers. Also current DNA databases consist of profiles comprising STR loci and therefore SNPs cannot be used in that context. At the same time it is possible to analyse hundreds of SNP loci and, due to their structure, the amplicon size can be much smaller, typically less than 100 bp. This allows the detection of DNA templates that are highly degraded and may generate data when standard STR typing fails to generate a result. A comparison between STR and SNP markers is shown in Table 12.1. In the foreseeable future, STRs will be the most commonly used genetic polymor- phism analysed. They are tried and tested in most judicial systems and also form the basis of most forensic DNA databases. Even so, the use of SNPs in forensic genetics is likely to increase in the coming years and may at some point in the future replace the

analysis of STR polymorphisms. The application of SNPs to specialized applications, for example, SNP based blood grouping [31, 39] and molecular autopsy (looking for mutations that can explain sudden death [40, 41]), is likely to become more widespread.

Further reading
Kwok, P.Y. (2001) Methods for genotyping single nucleotide polymorphisms. Annual Review of Genomics and Human Genetics 2, 235-258.

1. Miller, R.D., et al. (2005) High-density single-nucleotide polymorphism maps of the human genome. Genomics 86, 117-126.

2. Sachidanandam, R., et al. (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928-933.

3.Thorisson, G.A., and Stein, L.D. (2003) The SNP Consortium website: past, present and future. Nucleic Acids Research 31, 124-127.

4.Brookes, A.J. (1999) The essence of SNPs. Gene 234, 177-186.

5.Collins, F.S., et al. (2004) Finishing the euchromatic sequence of the human genome. Nature 431,931-945.

6.Gill,P.(2001) An as sessment of the utility of single nucleotide polymorphisms(SNPs) for forensic purposes. International Journal of Legal Medicine 114, 204-210.

7.Krawczak, M. (2001) Forensic evaluation of Y-STR haplotype matches: a comment. Forensic Science International 118, 114-115.

8.Danna, K., and Nathans, D. (1971) Studies of Sv40 DNA.1. Specific cleavage of Simian Virus 40 DNA by restriction endonuclease of hemophilus influenzae. Proceedings of the National Academy of Sciences of the United States of America 68, 2913-2917.

9.Denaro, M., et al. (1981) Ethnic variation in Hpa-I endonuclease cleavage patterns of human mitochondrial-DNA. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 78, 5768-5772.

10.Riehn, R., et al. (2005) Restriction mapping in nanofluidic devices. Proceedings of the National Academy of Sciences of the United States of America 102, 10012-10016.

11.Sanger, F., et al. (1977) DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74, 5463-5467.

12.Rosenblum, B.B., et al. (1997) New dye-labeled terminators for improved DNA sequencing patterns. Nucleic Acids Research 25, 4500-4504.

13.Prober, J.M., et al. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238, 336-341.

14.Albert, J., et al. (1993) Forensic evidence by DNA sequencing. Nature 361, 595-596.

15.Metzker, M.L., et al. (2002) Molecular evidence of HIV-1 transmission in a criminal case. Pro- ceedings of the National Academy of Sciences of the United States of America 99, 14292-14297.

16.Kwok, P.Y. (2001) Methods for genotyping single nucleotide polymorphisms. Annual Review of Genomics and Human Genetics 2, 235-258.

17.Syvanen, A.C., et al. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein-E. Genomics 8, 684-692.

18.Sanchez, J.J., et al. (2006) A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis 27, 1713-1724.

19.Ronaghi, M., et al. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363-365.

20.Ronaghi, M., et al. (1996) Real-time DNA sequencing using detection of pyrophosphate release. Analytical Biochemistry 242, 84-89.

21.Shumaker, J.M., et al. (1996) Mutation detection by solid phase primer extension. Human Mu- tation 7, 346-354.

22.Fan, J.B., et al. (2000) Parallel genotyping of human SNPs using generic high-density oligonu-cleotide tag arrays. Genome Research 10, 853-860.

23.Pastinen, T., et al. (2000) A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays. Genome Research 10, 1031-1042.

24.Saiki, R.K., et al. (1986) Analysis of enzymatically amplified beta-globin and Hla-Dq-Alpha DNA with allele-specific oligonucleotide probes. Nature 324, 163-166.

25.Livak, K.J., et al. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting Pcr product and nucleic-acid hybridization. Pcr- Methods and Applications 4, 357-362.

26.Lareu, M., et al. (2001) The use of the LightCycler for the detection of Y chromosome SNPs. Forensic Science International 118, 163-168.

27.Tyagi, S., et al. (1998) Multicolor molecular beacons for allele discrimination. Nature Biotech- nology 16, 49-53.

28.Tyagi, S., and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology 14, 303-308.

29.Wang, D.G., et al. (1998) Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the human genome. Science 280, 1077-1082.

30.Dixon, L.A., et al. (2005) Validation of a 21-locus autosomal SNP multiplex for forensic identi- fication purposes. Forensic Science International 154, 62-77.

31.Inagaki, S., et al. (2004) A new 39-plex analysis method for SNPs including 15 blood group loci. Forensic Science International 144, 45-57.

32.Dixon, L.A., et al. (2006) Analysis of artificially degraded DNA using STRs and SNPs – results of a collaborative European (EDNAP) exercise. Forensic Science International 164, 33-44.

33.Brion, M., et al. (2005) Introduction of an single nucleodite polymorphism-based ‘Major Y- chromosome haplogroup typing kit’ suitable for predicting the geographical origin of male lin- eages. Electrophoresis 26, 4411-4420.

34.Wetton, J.H., et al. (2005) Inferring the population of origin of DNA evidence within the UK by allele-specific hybridization of Y-SNPs. Forensic Science International 152, 45-53.

35.Frudakis, T., et al. (2003) A classifier for the SNP-based inference of ancestry. Journal of Forensic Sciences 48, 771-782.

36.Bastiaens, M., et al. (2001) The melanocortin-1-receptor gene is the major freckle gene. Human Molecular Genetics 10, 1701-1708.

37.Duffy,D.L.,etal.(2007)Athree-single-nucleotidepolymorphismhaplotypeinIntron1ofOCA2 explains most human eye-color variation. American Journal of Human genetics 80, 241-252.

38.Grimes, E.A., et al. (2001) Sequence polymorphism in the human melanocortin 1 receptor gene as an indicator of the red hair phenotype. Forensic Science International 122, 124-129.

39.Doi, Y., et al. (2004) A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples. Legal Medicine 6, 213-223.

40.Ackerman, M.J., et al. (2001) Molecular autopsy of sudden unexplained death in the young. American Journal of Forensic Medicine and Pathology 22, 105-111.

41.Levo, A., et al. (2003) Post-mortem SNP analysis of CYP2D6 gene reveals correlation between genotype and opioid drug (tramadol) metabolite ratios in blood. Forensic Science International 135,9-15.

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