Comparison of PrASE and Pyrosequencing for SNP Genotyping.
BACKGROUND
There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.


RESULTS
The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated.


CONCLUSIONS
The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.
Käller
Max
M
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE--106 91 Stockholm, Sweden. maxk@biotech.kth.se
Hultin
Emilie
E
Holmberg
Kristina
K
Persson
Marie-Louise
ML
Odeberg
Jacob
J
Lundeberg
Joakim
J
Ahmadian
Afshin
A
D000483:Alleles; D005838:Genotype; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*