Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices

Annika Ahlford, Bastian Kjeldsen, Jakob Reimers, Anders Lundmark, Massimo Romani, Anders Wolff, Ann-Christine Syvänen, Monica Brivio

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single ucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.
    Original languageEnglish
    JournalAnalyst
    Volume135
    Issue number9
    Pages (from-to)2377-2385
    ISSN0003-2654
    DOIs
    Publication statusPublished - 2010

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