Biomedical subjects

Emilie Hultin

Publications and source records attributed to Emilie Hultin.

3 recordsLinked to original sources

PubMed2006

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. CONCLUSION: 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.

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PubMed2005

Competitive enzymatic reaction to control allele-specific extensions.

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5' end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

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PubMed2005

Tag-array based HPV genotyping by competitive hybridization and extension.

A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (<2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

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