Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices.
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 nucleotide 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.
Ahlford
Annika
A
Department of Medical Sciences, Molecular Medicine, Uppsala University, University Hospital, Entrance70, SE-75185 Uppsala, Sweden.
Kjeldsen
Bastian
B
Reimers
Jakob
J
Lundmark
Anders
A
Romani
Massimo
M
Wolff
Anders
A
Syvänen
Ann-Christine
AC
Brivio
Monica
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005612:Freeze Drying; D005838:Genotype; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D005456:Fluorescent Dyes; D016159:Tumor Suppressor Protein p53; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D000469:Alkaline Phosphatase