A solid-phase method for preparing human DNA from urine for diagnostic purposes.
OBJECTIVE
To develop a simple method using paramagnetic beads for isolation of human DNA from small volumes of urine. The method should be amendable for automation. The purified DNA is intended to be used in downstream diagnostics and screening studies using nucleic acid amplification techniques.


METHODS
Unspecific capture of cells present in urine to magnetic particles, lysis and subsequent binding of the DNA to the same bead surface.


RESULTS
DNA isolated using the method could be used as template for sensitive real-time PCR and end-point PCR using primers targeted to the GAPDH, K-ras, DD3 and p53 genes. Compared to silica spin column-based extraction, the method showed equal or higher DNA yields. The method performed reliably when automated using a liquid handling robot equipped with a magnetic workstation.


CONCLUSIONS
The method generates purified DNA free from inhibitors, applicable for sensitive applications such as real-time PCR, genotyping, and for sequence variant analysis. The use of magnetic beads allows for automation, reducing hands-on time and creating a high throughput and reproducible protocol for the purpose of large-scale screening and diagnostics.
Siddiqui
Huma
H
Department of Biology, Centre for Ecological and Evolutionary Synthesis, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway.
Nederbragt
Alexander J
AJ
Jakobsen
Kjetill S
KS
D000328:Adult; D001331:Automation; D004247:DNA / Q000302:isolation & purification / Q000652:urine*; D019937:Diagnostic Techniques and Procedures; D005260:Female; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D008280:Magnetics; D008297:Male; D008863:Microspheres; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D052616:Solid Phase Extraction / Q000379:methods*; D013499:Surface Properties; D013997:Time Factors
D011933:Reagent Kits, Diagnostic; D004247:DNA