Either the standard phenol-chloroform extraction procedure (Blin and Stafford 1976) or the Autopure LS automated DNA purification instrument (Gentra Systems, Minneapolis, USA) was used to extract DNA from peripheral blood. The German DNA samples were extracted by salting out procedures.
5.2.2 Genotyping
5.2.2.1 Microsatellite markers
In study III the 72 families were genotyped using eight microsatellite markers surrounding the migraine candidate genes INSR and CACNA1A on 19p13. The markers covered a region of 11.4 Mb with a mean distance between markers of 1.6 Mb. The marker and gene positions based on the UCSC database (http://genome.ucsc.edu; Karolchik et al. 2003) are shown in Figure 5.
Figure 5. Locations of the eight genotyped microsatellite markers surrounding the INSR and CACNA1A genes on chromosome 19p13. (Chr, chromosome; Mb, megabase)
The genome-wide linkage analysis on the 36 families (study IV) was performed with the ABI Prism® Linkage Mapping Set v2.5 MD consisting of 400 microsatellite markers in a map with a resolution of approximately 10 cM (Applied Biosystems, Foster City, CA, USA). The
genetic map positions were based on the recombination rates derived from the Icelandic population (Kong et al. 2002).
5.2.2.2 Microsatellite genotyping
The repeat sequences of di- tri- or tetranucleotides were amplified by PCR (polymerase chain reaction, Mullis and Faloona 1987) using fluorescence-labelled oligos according to the manufacturer’s protocol. In study III DNA fragments were separated by capillary electrophoresis using the ABI3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA), called with GeneScan Software (Applied Biosystems) and analyzed with Genotyper Software (Applied Biosystems). In study IV the ABI3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) was used to separate the PCR fragments that were called with the ABI3730 data collection software and analyzed with the ABI Genemapper software package (Applied Biosystems). Genotypes of the CEPH (Centre d'Etudes du Polymorphisme Humain) individuals were used as controls to evaluate the performance of genotyping reactions and to standardize the allele calling.
All genotypes were verified by human inspection. Incompatibilities in Mendelian inheritance were controlled using the PedCheck program (O’Connell and Weeks, 1998). Genotype mistyping analysis was performed with the SimWalk2 program to reveal inconsistencies in genotypes based on allele frequencies, marker order map, phenotype model and pedigree structures (Sobel and Lange, 1996; Sobel et al. 2002). In mistyping analysis an overall error rate of 2.5% was used and all genotypes indicated with a probability of mistyping were rejected.
5.2.2.3 SNP markers
In study I a total of 33 SNPs were genotyped covering both the gene and flanking regions of the endothelin1 and its receptor A and B genes. The Haploview program (Barrett et al. 2005) was used to identify the haplotype tagging SNPs based on genotypes of Caucasian individuals in the public database of the International HapMap Project (http://www.hapmap.org/; The International HapMap Consortium 2003). If the designing of primers for tagging SNP failed, a
nearby SNP in as high LD (r2) as possible was selected to compensate for the original tagging SNP. The additional synonymous or non-synonymous SNPs from gene coding regions and template sequences were obtained from the UCSC Genome Browser (http://genome.ucsc.edu/, Karolchik et al. 2003). The Positions and LD structure of analyzed SNPs are shown in Figure 1 (study I).
For study II 96 SNPs were genotyped including the 32 SNPs from study I. The rest of the SNPs were selected either from the public databases mentioned above or from the Seattle SNP database (http://pga.gs.washington.edu/).
5.2.2.4 SNP genotyping
The SNP genotyping (studies I and II) was performed with the homogenous Mass Extension Mass ARRAY genotyping system (Sequenom®, San Diego, CA, USA). The allele identification is based on the mass differences between two alleles of a SNP (Leushner et al.
2000). Figure 6 shows an overview of the primer detection reaction for the A/T SNP polymorphism: A known region of genomic DNA is amplified and then used as a template for a primer extension reaction. A detection primer is designed to anneal adjacent to a SNP site (A/T). The DNA polymerase enzyme mediates the extension of the detection primer with a single thymidine dideoxynucleotide triphosphate (ddTTP). To gain at least 50 Da difference in mass of extension products a primer for allele T is firstly extended with an adenosine deoxynucleotide (dATP) and then terminated by adding a guanine dideoxynucleotide triphosphate (ddGTP).
Figure 6. Overview on the SNP identification by using the homogenous Mass Extension Mass ARRAY.
For the Sequenom genotyping system the AssayDesign 2.0.7.0 software (Sequenom) was used to design multiplexes of 4–6 SNPs for the PCR and extension reactions. PCR and extension primers were purchased from Proligo France SAS (Paris, France) and Metabion International AG (Martinsried, Germany), respectively. The extension reaction was performed with 0.6 U/reaction using either ThermoSequenase® (GE Healthcare, Chalfont St. Giles, UK) or TERMIPol® DNA polymerase (Solis Biodyne OÜ, Tartu, Estonia). Genotyping of each SNP was first evaluated in a single-plex reaction for optimal primer concentrations in the sample set of 31 anonymous blood donors. In the second optimization step SNPs were genotyped in a multi-plex reaction of 4–6 SNPs to detect possible primer-primer interactions and assess the optimal primer concentrations between multiple primer pairs before sample genotyping. Aside from optimization modifications, the multiplex PCR reactions were performed according to the manufacturer’s instructions.
Extension products spotted on microchips were detected with a real-time matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The genotypes were called by the SpectroCaller software (Sequenom). All genotypes were verified by human inspection with the Typer 3.3 software (Sequenom). The quality of genotypes was
inspected by the KariOTyper in-house web interface (Study II). The SPSS (version 12.0.1) software was used to calculate the numeric parameters in order to compare the performance of two DNA polymerases in study II. In study I genotype distributions in the whole sample and for cases and controls were tested separately for the Hardy-Weinberg equilibrium (HWE) by the χ2 method implemented in the PLINK 1.0 program in order to reject the suspicious markers (Purcell et al. 2007). In the HWE test a p-value <0.001 was used as an exclusion threshold for SNPs (http://www.hapmap.org/downloads/data-handling_protocols.html).