We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3 and 5 ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed...

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled o...

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase (Pol ) replicated DNA at a rate of 40–60 nt/s. When downstream double-stranded DNA was encountered, Pol paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 n...

Fig. S1. Results of polymerase chain reaction (PCR) using universal primers targeting mycobacterium DNA: hsp65 short (hsp65S; 441 bp), long (hsp65L; ca.770 bp), rpoB short (rpoBS; ca.330bp), long (rpoBL; ca.450bp) genes and the 16S-23S spacer region (internal transcribed spacer (ITS); ca. 350 bp). Clear strong bands with hsp65L, rpoBS, and ITS primers proved the existence of mycobacteria DNA. I...

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and therm...

BACKGROUND Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing te...

Telomerase is a specialized reverse transcriptase that contains an integral RNA subunit including a short template sequence. It extends telomeric 3' overhangs and chromosome breakpoints by catalyzing reiterative copying of this internal template into single-stranded telomeric DNA repeats. Here we report for the first time that in vitro the ciliate Tetrahymena telomerase can efficiently extend v...

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. Th...

A method, termed hierarchical oligonucleotide primer extension (HOPE), is developed for quantitative, multiplexing detection of DNA targets present in PCR-amplified community 16S rRNA genes. It involves strand extension reaction and multiple oligonucleotide primers modified with different lengths of polyA at the 5' end and targeting 16S rRNA genes at different phylogenetic specificities. On ann...

Aims-To evaluate factors which ameliorate false positive artefacts with direct in situ PCR using labelled dNTPs; to investigate the use of labelled primers to overcome this artefact whilst maintaining sensitivity.Methods-Sections of measles (RNA virus) infected Vero cells with cytoplasmic signal or cytomegalovirus (DNA virus) infected fibroblasts with nuclear signal were collected. In situ PCR ...