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Technical Briefs |
1 Department of Biotechnology, The Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden;2 Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden
aaddress correspondence to this author at: Department of Biotechnology, The Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden; fax 46-8-5537-8481, e-mail joakim.lundeberg{at}biotech.kth.se
The red hair color phenotype is associated with an increased risk of developing cutaneous malignant melanoma (1) and has been linked to certain variants of the gene for melanocortin-1 receptor (MC1R) (2)(3)(4). The function of the receptor is to mediate melanin production via binding of its ligand, 
-melanocyte-stimulating hormone, and/or adrenocorticotropic hormone (5). The presence of some MC1R variants fails to shift the production from red/yellow pigment (pheomelanin) to black/brown pigment (eumelanin), causing less efficient protection against ultraviolet radiation. The MC1R gene is highly polymorphic, and many identified variants have been associated with an increased risk of developing malignant melanoma (6)(7). Some of these variants have also been proposed to synergize with mutations of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A) to enhance the melanoma risk further (8)(9).
There are many novel technologies for the analysis of single-nucleotide polymorphisms (SNPs) and mutations. We have previously described an approach to increase the specificity of the allele-specific extension (ASE) technology for SNP genotyping and mutation detection (10). Interestingly, this allows typing of several SNPs that were not possible to analyze by unmodified ASE. The ASE technology uses the ability of DNA polymerases to distinguish matched and mismatched 3' termini of primers and thus to extend only completely matched primers. However, several reports have shown that some 3'-termini mismatches can be elongated and give false-positive results (11). This problem can be circumvented by exploiting the fact that the mismatched primers have slower reaction kinetics. The elongation can be terminated by protease (proteinase K) degradation of the polymerase before any incorrect insertions are made (10). Protease-mediated allele-specific extension (PrASE) is a flexible assay and can be performed at lower temperatures using thermolabile enzymes, such as the Klenow fragment, or at increased temperatures using thermostable DNA polymerases, such as Taq polymerase.
An assay based on PrASE was set up for screening of the MC1R and CDKN2A polymorphisms suggested to be present in the Swedish population. The PrASE assay used is outlined in Fig. 1
. PCR products of MC1R and CDKN2A were pooled and immobilized on magnetic beads by biotin-streptavidin binding. This facilitates the automation of 48 PrASE reactions in parallel with a magnet-equipped pipetting robot. Cy5-labeled deoxynucleotide triphosphates were used to allow fluorescence detection of the extension products by use of a microarray system of spotted tag oligonucleotides complementary to signature tags on the extension primers. A subarray system facilitated the analysis of 48 samples on 1 slide. Genotyping calls were made by cluster analysis of the fluorescence signals.
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A total of 21 MC1R variants were chosen from initial Sanger sequencing results and previously published studies (Table 1
). Two variants in CDKN2A were also chosen: the codon 113 arginine insertion founder mutation (ins113R), which is the most common CDKN2A alteration in Swedish melanoma families (12)(13), and the A148T amino acid change, because it has been proposed to be associated with increased risk of development of malignant melanoma (14)(15)(16).
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The investigation was performed on DNA isolated from peripheral lymphocytes from 42 healthy controls and 50 patients with sporadic cutaneous malignant melanomas. Primers and PCR protocols can be found in the Data Supplement accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue12/.
Single-fragment PCR products of MC1R were sequenced directly by standard protocols for ABI BigDye Terminator chemistry (Applied Biosystems). In total, 7 sequencing primers were used (Table 1
in the online Data Supplement), giving overlapping bidirectional sequences for the coding region and monodirectional sequences for promoter sequence starting at the –275 position.
Generic tag arrays were prepared as described previously (10)(17) with 48 oligonucleotide tags functioning as capture probes (MWG-Biotech AG). The oligonucleotides were spotted with a Q-array (Genetix) on Code Link activated slides (Amersham Biosciences). This was followed by post coupling as outlined by the manufacturer. The oligonucleotides were printed in triplicate in 48 identical arrays that were separated during hybridization by a reusable silicone mask, allowing simultaneous hybridization of 48 samples on 1 slide.
The steps for PCR product immobilization, washing, annealing of 3'-terminus allele-specific primers (Table 2 in the online Data Supplement), and multiplex allele-specific extensions were automated by the use of a Magnatrix 1200 pipetting robot system capable of handling magnetic beads (Magnetic Biosolutions). However, in some regions of the PCR product the polymorphic positions studies are located in very close proximity, which causes the individual extension primers to overlap. This could lead to conflicts in both hybridization and primer extension; thus, the extension primers were separated into 2 sets (indicated in Fig. 1
). Each set was annealed and extended separately but pooled before hybridization to the microarray. For each reaction, 20 µL of PCR product from MC1R was used with an addition of 5 µL of a shorter MC1R fragment and 6 µL of the CDKN2A fragment in set 1. Streptavidin-coated super paramagnetic beads (Dynabeads M280; Dynal) were used to immobilize the biotinylated PCR products. Single-stranded DNA was obtained before multiplex annealing of the extension primers, and after subsequent removal of surplus primers, the beads were resuspended in 20 µL of 1x annealing buffer [10 mmol/L Tris-acetate (pH 7.75), 2 mmol/L magnesium acetate].
The PrASE reaction was performed at 65 °C by first adding 20 µL of a solution containing 10 U of Taq DNA polymerase, 1x extension buffer [42.5 mmol/L Tris-HCl (pH 8), 5 mmol/L MgCl2, and 1 mmol/L dithiothreitol], and 0.25 g/L bovine serum albumin. Following this, 20 µL of a mixture containing 1.5 µmol/L of each deoxynucleotide triphosphate (50% Cy5-labeled dCTP and dTTP; Amersham Biosciences), 2x extension buffer, 0.5 g/L bovine serum albumin, and 20 µg of proteinase K was then added to initiate the extension by the polymerase and simultaneously terminate the extension by degradation of the polymerase by the protease. After polymerization, the immobilized DNA was washed with 1x annealing buffer. The 2 sets of separately extended primers were then pooled. Strand-specific alkali elution of the extended primers was performed, and 2x hybridization buffer (10x saline sodium citrate–4 g/L sodium dodecyl sulfate) was added to a total volume of 32 µL. The extension primers each contained specific signature tags that were hybridized to their complementary probes on the generic tag arrays for 1 h. Sample genotyping was performed by automatic cluster analysis of the fluorescence signals by use of a Microsoft Excel script (10).
A total of 2116 genotypes in 92 samples were examined by both Sanger DNA sequencing and PrASE, and 100% concordance was achieved. It should be mentioned that additional variants were not detected by Sanger DNA sequencing. The allele frequencies of the investigated polymorphisms are listed in Table 1
. Of the 23 MC1R and CDKN2A polymorphisms, 16 were found in the examined sample material. For 8 of the polymorphisms, double mutants were found. Note that the samples were selected from a larger pool of both healthy controls and patients with sporadic malignant melanomas from a Swedish population and were chosen to contain many different variants. Hence, the allele frequencies cannot be compared with those of previous reports.
The PrASE assay for detection of common variants in MC1R and CDKN2A was as accurate as Sanger DNA sequencing. However, PrASE technology provides substantial reductions in labor, time, and cost. The total cost of the PrASE genotyping in this study was estimated to be less than $2.00 (US), including all reagents and primers, per individual (9 cents per SNP). Genotyping of MC1R and CDKN2A by Sanger DNA sequencing (with BigDye chemistry and use of capillary electrophoresis instruments) would require $9.00 (US) per individual. In addition, because the PrASE assay is robotized and analysis of the results is highly automated and printed in a convenient format, the total genotyping procedure is estimated to take 4 h. Sanger sequencing is considerably more time-consuming because 7 reactions are needed to cover the MC1R and CDKN2A genes and the vast majority of the steps, including sequencing reactions and clean ups, sequence editing, and genotype recording, are performed manually. In addition, most laboratories are equipped with slab-gel sequencing instruments, and thus the time for gel preparation and sample loading should also be considered. The use of a tag-microarray detection system makes the choice of variants to be examined by PrASE very flexible because only a biotinylated PCR product and 2 extension primers must be designed to include a variant in the assay. The current design allows simultaneous analysis of 24 variants, which can be chosen from as many genes. However, the flexibility of the arrays and the PrASE assay allows increased throughput for parallel analysis of at least 75 variable loci with genotyping costs similar to those for the analysis of 24 polymorphic positions.
In conclusion, the flexibility and multiplexing features of the PrASE assay provide an accurate and convenient alternative to microarray-based technologies for analysis of polymorphic positions in 1 or several different genes.
Acknowledgments
This work was supported by grants from the Swedish Research Council, the Swedish Medical Research Council, the Swedish Cancer Society, the Knut and Alice Wallenberg Foundation, the Cancer Society of Stockholm, the King Gustav V Jubilee Fund, the Karolinska Institute Research Funds, and the Swedish Radiation Protection Institute.
References
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