Analyzing rare DNA and RNA molecules in limited sample sizes, such as liquid biopsies and single cells, often requires preamplification, which makes downstream analyses particularly sensitive to polymerase chain reaction (PCR) generated contamination. Herein, we assessed the feasibility of performing Cod uracil-DNA N-glycosylase (Cod UNG) treatment in combination with targeted preamplification, using deoxyuridine triphosphate (dUTP) to eliminate carry-over DNA. Cod UNG can be completely and irreversibly heat inactivated, a prerequisite in preamplification methods, where any loss of amplicons is detrimental to subsequent quantification. Using 96 target assays and quantitative real-time PCR, we show that replacement of deoxythymidine triphosphate (dTTP) with dUTP in the preamplification reaction mix results in comparable dynamic range, reproducibility, and sensitivity. Moreover, Cod UNG essentially removes all uracil-containing template of most assays, regardless of initial concentration, without affecting downstream analyses. Finally, we demonstrate that the use of Cod UNG and dUTP in targeted preamplification can easily be included in the workflow for single-cell gene expression profiling. In summary, Cod UNG treatment in combination with targeted preamplification using dUTP provides a simple and efficient solution to eliminate carry-over contamination and the generation of false positives and inaccurate quantification.

• MeSH
• analýza jednotlivých buněk MeSH
• deoxyuracilnukleotidy metabolismus   MeSH
• Gadus morhua metabolismus   MeSH
• kontaminace DNA * MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese MeSH
• uracil-DNA-glykosidasa metabolismus   MeSH
• uracil metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• mezinárodní spolupráce MeSH
• poskytování zdravotní péče MeSH
• soudní lékařství MeSH
• zákonodárství jako téma MeSH
• zákonodárství lékařské MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Právo
• NLK Obory
• právo, zákonodárství
• lékařství