Available Human Snp Markers

Large national and international efforts have been underway over the past few years to catalog human variation found in the form of SNP markers. The SNP Consortium (TSC) was established in the spring of 1999 to create a high-density

SNP map of the human genome. The TSC is a collaboration between major pharmaceutical companies, the Wellcome Trust (the world's largest medical research charity), and five leading academic and genome sequencing centers (Holden 2002; see http://snp.cshl.org). The TSC effort has produced several million mapped and characterized human SNP markers that have been entered into public databases including dbSNP housed at the National Institute of Health's National Center for Biotechnology Information (Sherry et al. 2001; see http://www.ncbi.nlm.nih.gov/SNP). Additionally, the International HapMap project is a follow-on to the Human Genome Project (see Chapter 2) and includes a plan to type 270 individuals from African, European, and Asian populations with approximately one million SNPs (Gibbs et al. 2003; see http://www.hapmap.org/). With these large ventures on-going around the world, there will be no shortage of available SNP markers and accompanying population data.

Several members of the European forensic DNA typing community launched a project in 2003 known as SNPforID that is developing SNP assays to directly aid forensic DNA analysis (Phillips et al. 2004). This group is endeavoring to develop highly multiplexed SNP assays using unlinked loci that are well spread throughout the human genome. Population data is also being gathered to measure SNP allele frequencies in various groups of interest.


Table 8.2

SNP analysis techniques.

A number of SNP typing methods are available, each with their own strengths and weaknesses. Several reviews of SNP typing technologies have been published and can be consulted for a more in-depth view of methodologies than will be presented here (see Gut 2001, Kwok 2001, Syvânen 2001). A summary and brief description of SNP analysis techniques are listed in Table 8.2.




Reverse dot blot or linear arrays

Genetic bit analysis

Direct sequencing Denaturing HPLC

A series of allele-specific probes are attached to a nylon test strip at separate sites; biotinylated PCR products hybridize to their complementary probes and are then detected with a colorimetric reaction and evaluated visually

Primer extension with ddNTPs is detected with a colorimetric assay in a 96-well format

Nikiforov et al. (1994)

PCR products are sequenced and compared to reveal SNP sites Kwok et al. (1994)

Two PCR products are mixed and injected on an ion-paired reversed-phase HPLC; single base differences in the two amplicons will be revealed by extra heteroduplex peaks

Hecker et al. (1999)





TaqMan 5' nuclease assay

Fluorescence polarization

Mass spectrometry

High-density arrays (Affymetrix chip)

Electronic dot blot (Nanogen chip)

Molecular beacons

Oligonucleotide ligation assay (OLA)

Tm-shift genotyping


Allele-specific hybridization (Luminex™ 100)

Minisequencing (SNaPshot™ assay)

A fluorescent probe consisting of reporter and quencher dyes is added to a PCR reaction; amplification of a probe-specific product causes cleavage of the probe and generates an increase in fluorescence

Primer extension across the SNP site with dye-labeled ddNTPs; monitoring changes in fluorescence polarization reveals which dye is bound to the primer

Primer extension across the SNP site with ddNTPs; mass difference between the primer and extension product is measured to reveal nucleotide(s) present

Thousands of oligonucleotide probes are represented at specific locations on a microchip array; fluorescently labeled PCR products hybridize to complementary probes to reveal SNPs

Potential SNP alleles are placed at discrete locations on a microchip array; an electric field at each point in the array is used to control hybridization stringency

Hairpin stem on oligonucleotide probe keeps fluorophore and its quencher in contact until hybridization to DNA target, which results in fluorescence

Colorimetric assay in microtiter 96-well format involving ligation of two probes if the complementary base is present

Allelic-specific PCR is performed with a GC-tail attached to one of the forward allele-specific primers; amplified allele with GC-tailed primer will exhibit a melting curve at a higher temperature

Sequencing by synthesis of 20-30 nucleotides beyond primer site; dNTPs are added in a specific order and those incorporated result in release of pyrophosphate and light through an enzyme cascade

Dye-labeled PCR products hybridize to oligonucleotide probes (representing the various SNP types) attached to as many as 100 different colored beads; each bead is interrogated to determine its color and whether or not a PCR product is attached as the beads pass two lasers in a flow cytometer

Allele-specific primer extension across the SNP site with fluorescently labeled ddNTPs; mobility modifying tails can be added to the 5'-end of each primer in order to spatially separate them during electrophoresis

Livak (1999)

Giesendorf et al. (1998)

Delahunty et al. (1996)

Germer and Higuchi (1999)

Anmadian et al. (2000), Andreasson et al. (2002)

SNPstream® UHT

High-tech version of Genetic bit analysis with a 384-well tag array and 12plex PCR

Some of the primary SNP typing methods that have received attention in the forensic community include pyrosequencing (Andreasson et al. 2002), TaqMan (Lareu et al. 2001), Luminex (Budowle et al. 2004), and minisequencing or SNaPshot™ (Tully et al. 1996, Sanchez et al. 2003). One of the important characteristics of a SNP assay is its ability to examine multiple markers simultaneously since SNPs are not as variable as STRs and typically a limited amount of DNA template is available in forensic casework. While pyrosequencing and TaqMan assays are limited in their multiplexing capabilities, Luminex and minisequencing assays enable multiplexed analysis of a dozen or more SNP markers simultaneously. Minisequencing is now a viable SNP typing option with the availability of the SNaPshot™ kit (Applied Biosystems, Foster City, CA) and multi-colored fluorescent detection electrophoresis instrumentation. In the next section, we go into detail about the SNaPshot assay.

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