PCR-generated padlock probes detect single nucleotide variation in genomic DNA

Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5′- and 3′-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.     

Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.     

Species identification in meat by using PCR-generated satellite probes

A convenient DNA-based identification system is described for testing the species origin of meat samples. Probes are generated by PCR with primers binding to species-specific satellite DNA and hybridized to DNA purified from meat. This method is more robust and versatile than methods based on oligonucleotide hybridization. With the exception of a slight cross-reaction of mutton and beef, each probe only recognized the species from which it was derived. Purifying the DNA with a DNA-binding resin improved the sensitivity. Admixtures of 0.1–0.5% can be detected in raw meat and 0.5–5% in autoclaved meat samples. The method can be adapted to detect any eukaryotic species for which species-specific DNA sequences are available. This method has proven its value in the routine inspection of meat samples by revealing more cases of deliberate or accidental species substitution and admixture than conventional techniques. Received 12 January 1998/ Accepted in revised form 15 June 1998     

Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations

A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.     

Synthesis of DNA polymerase by in vitro translation of calf RNA

Synthesis of alpha-polymerase in translation mixtures containing calf thymus poly(A+) RNA was examined by activity gel analysis and by immuno-binding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that a DNA polymerase catalytic polypeptide of Mr = approximately 120,000 had been synthesized. Immunobinding experiments indicated that an immunoreactive polypeptide of about the same size had been formed in vitro. Sucrose gradient centrifugation of calf thymus total RNA revealed that mRNA encoding the approximately 120,000-Mr DNA polymerase polypeptide sedimented at about 16S. This approximately 120,000-Mr catalytic polypeptide corresponds in size to an alpha-polymerase catalytic polypeptide found earlier in crude extracts of calf cells.