Mark S. Berninger

Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions

Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.

Bioassay for specific DNA sequences using a non-radioactive probe.

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.

16 – Ligation-Activated Transcription Amplification: Amplification and Detection of Human Papillomaviruses

This chapter describes an isothermal method for amplification of nucleic acids based on ligation of a promoter sequence to a target nucleic acid, resulting in target amplification through a transcribed RNA intermediate. This target amplification methodology has been combined with solution hybridization based enzymatic detection of amplified target nucleic acid to produce a highly sensitive method for the detection of nucleic acids. Application of these technologies to detection of human papillomaviruses (HPV) is described. The widespread utility of amplification in molecular biology and molecular diagnostic procedures has stimulated the development of other nucleic acid amplification methods. Some of these methods are dependent on the presence of a DNA target and amplify a target sequence; other methods are based on replicatable probes that are amplified after hybridization to reveal the presence of the probe. The ligation-activated transcription reaction is performed under isothermal conditions without thermocycling. The reaction is capable of using RNA or DNA as the starting template and produces both single-stranded RNA and DNA amplification products.