We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma. Results: The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan ® assay (interassay CVs, 5%-20% vs 7%-

Aim: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. Methods and Results: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5′‐TTCGAA‐3′), Asp83/1II (5′‐GGCC‐3′), Asp90I (5′‐ACRYGT‐3′) and five isoschizomeric enzymes (5′‐ATCGAT‐3′). Aphanizomenon and Microcystis strains contained ApcTR183I (5′‐TGCGCA‐3′) and Msp199I (5′‐CCGG‐3′), respectively. Planktothrix strains possessed Psc2I (5′‐GAANNNNTTC‐3′), Psc27I and Psc28I (5′‐TTCGAA‐3′). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. Conclusions: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). Significance and Impact of the Study: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.

RmaI, an isoschizomer of MaeI (1) has been isolated from Rhodothermus marinus (2). RmaI recognizes the palindromic sequence 5'-CTAG-3'. Like its isoschizomer RmaI cleaves the sequence, C/TAG, generating 5'-protruding TA-dinucleotides.The recognition sequence of RmaI was determined using double digestions on pBR322and phiXI74 DNAs (figure 1, lanes 2-6 and 8-12). The cleavage patterns obtained were compared with computer-derived mapping data (3). The data predicts the sequence 5'-CTAG-3'. The sequence was also tested by digesting Lambda-and Ml3mpl8 DNAs with RmaI (figure 1, lanes 13 and 14). The fragments obtained matched the computer predicted fragments that would be produced when cleaving at 5'-CTAG-3'. RmaI has the following number of recognition sites on these commonly used DNAs: pUC19 (4), pBR322 (5), phiX174 (3), SV40 (12), Ml3mpl8 (5), T7 (60), Lambda (13) and Adeno2 (54).The cleavage site of RmaI was determined by cleavage of primed synthesis reaction (4). M13mpl9 DNA containing the recognition site of RmaI was used as a template. Using a M13 sequencing primer the DNA was sequenced according to Sanger et al. ( 5). In addition to the four standard reactions a fifth reaction was performed which was extended through the RmaI recognition site. The reaction was terminated by heat treatment and the product was cleaved with RmaI. The cleaved product was divided in two. The addition of Klenow to one part resulted in a band migrating with the A-band (figure 2, lane +). The cleaved product (figure 2, lane -) migrated with the C-band. Thus, the cleavage site for RmaI is: 5'-C/TA-G-3' 3'-G-AT/C-5'