Signet™ Y-SNP Genotyping Kits

Signet™ Genotyping Technology

Marligen’s Signet™ Y-SNP Genotyping kits use a simple hybridization-based assay (Figure 1).

Figure 1. Schematic diagram of the assay procedure.

Following multiplex PCR amplification and labeling via asymmetric PCR using a universal fluorescently labeled primer, the PCR products differentially hybridize to pairs of oligonucleotide probes complementary to each possible allele sequence that are covalently attached to spectrally distinguishable microspheres. Following hybridization, the signal is detected using a Luminex instrument. The genotype is determined from the ratio of signal obtained from each pair of beads.

These kits employ a two tiered strategy that efficiently detects 96 polymorphic markers in multiplexed PCR and detection reactions.

Samples are first analyzed with a screening multiplex (A-R) that determines the major haplotype group of each sample. Subsequently, samples are analyzed with one of the haplogroup-specific multiplexes (AB, CD, E, FGHI, J, KLMN, O1, O2, PQ, R1 or R2) to determine the precise haplotype of each sample. A schematic diagram of the strategy for genotyping is shown in Figure 2 below.

Figure 2. A schematic diagram of the two tiered strategy employed in the Y-SNP Genotyping Kits.

The product line is comprised of the Signet™ Y-SNP Genotyping Core Kit, which contains the common reagents needed for all of the individual genotyping kits (Labeling Mix, Exonuclease I, Hybridization Solution and Control DNAs, male and female), the Signet™ Y-SNP A-R Primary Screening Kit containing multiplexed PCR Mix and Bead Mix for classifying each sample at the root level, and eleven Signet™ Y-SNP Genotyping Kits that contain the PCR Mixes and the Bead Mixes for assigning the samples to specific haplogroups. The Primary Screening Kit and the Genotyping Kits are designed for use in conjunction with the Core Kit. Each of these kits provides reagents sufficient for analyzing 100 samples.

Marligen's Signet™ Y-SNP Genotyping Kits are configured to be used in conjunction with the Luminex® system. Data acquisition can be done with Luminex® Data Collector or with MiraiBio MasterPlex™ CT software. Data analysis can be done with MiraiBio MasterPlex™ GT or Microsoft Excel.

Marligen also offers Signet™ Genotyping Testing Services that include sample preparation, testing, and data analysis.

The table below details the compositions of the probe and primer multiplexes that are currently available. If additional markers are required, Marligen’s scientists will work with you to design, develop, and manufacture specific Custom SNP Genotyping Assays to meet your research needs.

Performance Characteristics

Figures 3 and 4 show an example of the results obtained upon testing a male DNA sample with the Signet™ Y-SNP Genotyping Kits as described in the schematic diagram shown in Figure 2. The sample was first tested with the Signet™ Y-SNP A-R Primary Screening Kit, and the resulting genotype was determined to be M9C/M45G/M175ins, a member of the KLMN haplogroup. The sample was subsequently tested with the Y-SNP KLMN Genotyping kit and was determined to be haplotype N3a (M214C/M231A/TatC/M178T).

Fig. 3. Plot of relative intensity for the alleles of a test DNA sample with the A-R multiplex. The sample type is M9C/M45G/M175ins, therefore, this sample needs to be typed further with the KLMN multiplex.

Fig. 4. Plot of relative intensity for the alleles of a test DNA sample with the KLMN multiplex. The sample has been typed as M214C/M231A/TatC/M178T, and its haplotype is N3a.

Genotyping results obtained with the Signet™ Y-SNP Genotyping Kits have been shown to have 100% concordance with sequencing. Very clean allelic discrimination is achieved, as is demonstrated in Figure 5, which shows an example of the data obtained for the M9 locus from 44 samples.

Fig. 5. Excellent allelic discrimination is achieved with the Signet™ Y-SNP Genotyping Kits. This figure shows Signet™ genotyping results at Y-SNP Allele M9 from 44 male DNA samples. The signal for each allele from 44 male DNA samples is plotted.