Biomedical subjects

D O Antson

Publications and source records attributed to D O Antson.

4 recordsLinked to original sources

PubMed2001

RNA-templated DNA ligation for transcript analysis.

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.

Bacteriophage T4↗

PubMed2001

More keys to padlock probes: mechanisms for high-throughput nucleic acid analysis.

With the impending availability of total information about nucleic acid sequences in humans and other organisms, tools to investigate these sequences on a large scale assume increasing importance. Methods currently in use, however, cannot offer the required combination of high-throughput, sensitivity and specificity of detection. Padlock probes, circularizing oligonucleotides, may provide a means to detect, distinguish, quantitate and also locate very large numbers of DNA or RNA sequences. Recent developments in areas such as the biochemistry of ligation and characterization of ligases, methods to replicate circularized probes and the development of assays based on these principles augment the potential of padlock probes.

Biotechnology↗

PubMed2000

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.

Adenosine Triphosphatases↗

PubMed2000

Enhanced detection and distinction of RNA by enzymatic probe ligation.

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe-target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.

Alleles↗