The multiplex SNaPshot detection of single-base extended probe primers was based on fluorescence and extended length detected by capillary electrophoresis on an ABI3130XL Sequencer (Applied Biosystems).
Taking the advantage of this method, two multiplex SNaPshot minisequencing assays were developed for simultaneous genotyping of twelve SNPs in the BCL11A gene and sixteen SNPs in the HBS1L-MYB intergenic region.
The multiplex SNaPshot reaction was performed in a final volume of 10 μL, containing 50 ng of each template, 5.0 μL of the SNaPshot multiplex ready reaction mix, and 4 μM of each SNaPshot primers for HPA-1, -2, -3, -4, -5, -6, -7, -9, and 10 μM of primers for HPA-8, -13, and -15.
Multiplex SNaPshot minisequencing is achieved by mixing primers of different lengths with the SNaPshot mix and the multiplex PCR template.
The treated PCR products were used as template in a multiplex SNaPshot minisequencing extension reaction containing a pool of polymorphism-specific primers.
On the PCR templates described above, these primers were elongated at the variation sites by fluorescently labelled ddNTPs in total reaction volumes of 5 μl, using the multiplex SNaPshot kit (Applied Biosystems, Foster City, USA).
B. Three genotypes of rs 7518660 performed by the multiplex SNaPshot technique.
After the successful detection of all polymorphisms with these two multiplex SNaPshot minisequencing assays, 25 genomic DNA β-thalassaemia patients were tested each carrying an unknown genotypic SNPs profile.
To verify the genotyping of the selected BCL11A and HBS1L-MYB polymorphisms obtained with the multiplex SNaPshot minisequencing assay we carried out Sanger DNA sequencing and/or PCR-RFLP analyses of 25 random β-thalassaemia samples.
The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations.
