Chelex 100

KRAS mutation tests were performed at the designated central laboratory of SMC as described previously [31 (link)]. Mutations in codons 12 and 13 of the KRAS gene were detected by direct sequencing of polymerase chain reaction (PCR) products amplified from DNA extracted from representative tumor tissue. BRAF V600E direct sequencing and KIT hotspot mutations were tested according to our previous work [32 (link)]. Briefly, tumor-rich areas (>80%) were extracted from paraffin–embedded tissue sections, and 10 4-μm-thick sections containing a representative portion of each tumor block were subjected to DNA isolation using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Deeply pigmented samples were incubated with Chelex-100 (Bio-Rad Laboratories) to prevent PCR inhibition by melanin [33 (link)]. Purified DNA was incubated for 10 min at room temperature with an equal volume of a 5% Chelex-100 solution equilibrated in Qiagen AE buffer, heated to 95°C for 2 min, and allowed to cool. The Chelex-100 resin was pelleted in a microfuge, and the supernatant DNA used for PCR reactions. PCR products were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster, CA, USA) with both forward and reverse sequence-specific primers. Sequence data were generated using the ABI PRISM 3100 DNA Analyzer (Applied Biosystems).

Ethical approval for two ongoing studies on glycophorins and malaria was granted by the Ethics Committee for Basic and Applied Sciences, College of Basic and Applied Sciences, University of Ghana (CPN: ECBAS 037/18–19), and the Noguchi Memorial Institute for Medical Research IRB, University of Ghana (CPN 004/11–12). Written informed consent was obtained from all the study participants or their parents/guardians in the case of the children. The assays developed were used to genotype DNA samples obtained from volunteers who had been enrolled in various ongoing studies in three areas of Ghana namely: Accra, Kintampo, and Hohoe.
Venous blood samples were collected and following curation of self-reported ethnicity to only include individuals of Ghanaian origin, comprised; Kintampo (n = 147), Hohoe (n = 43), and Accra (n = 203) (Figure 6, Table 5). Genomic DNA was extracted using the Qiagen QIAmp Blood Mini Kit or Chelex-100 as described for different batches of samples.^{28 (link)} The DNA samples were quantified using Picogreen as described above. The gDNA samples were diluted to 20 ng/µL and stored in 96-well PCR plates at −20°C until ready for genotyping. GYPB DEL1 and DEL2 genotyping assays were undertaken as given in Tables 3 and 4.