Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

Notes: After bioinformatics analysis of the Bacillus anthracis genome for potential vaccine targets, the 197 ORFs were selected for amplification. Using the genomic DNA of B. anthracis strain Vollum, all PCR products were generated using a 5’ primer incorporating the T7 promoter, the Kozak sequence, unique restriction enzyme sites and a start codon and a 3’ primer with a stop codon and three more unique restriction sites. The amplicons were then in vitro transcribed and translated using the TNT^® T7 Quick for PCR DNA system and radiolabeled with [³⁵S]methionine. The products were analyzed by SDS-PAGE and autoradiography. To assess the immunoreactivity of the translated products, the proteins were immunoprecipitated using anti-B. anthracis antisera. The seropositive candidate amplified ORFs were cloned into the pCI Mammalian Expression Vector using compatible RE sites. These vectors were introduced into ICR mice using a Helios gene gun system (0.5µg plasmid DNA on 1µm gold particles) and the mice immunized in 2 week intervals. (3515)

Notes: A region of the ORF from the APC gene from colorectal cancer and normal patients was isolated and amplified by PCR. The primers used incorporated transcription and translation sequence elements as well as N- and C-terminal FLAG and HA tags respectively. The PCR products were used as templates in TNT^® T7 Quick for PCR transcription/translation reactions.  Four different colored BODIPY-fluor-labeled lysines were added to the reactions to label the proteins in the TNT^® T7 Quick for PCR transcription/translation reactions. A subtractive precipitation strategy was used to purify full-length and truncated APC proteins based on the presences or absence of FLAG and HA tags. The partially purified proteins were run on SDS-PAGE gels and imaged with a Typhoon 9700 instrument to detect truncations.    (3026)

Notes: The sterol regulatory element-binding protein-1a (SREBP1a) was in vitro transcribed and translated from a plasmid template using TNT^® T7 Quick for PCR DNA. A control reaction was performed using Transcend™ tRNA to confirm that the 120KDa protein was expressed correctly.  Unlabeled SREBP1a was used in gel-shift assays with a labeled oligo representing the SRE motif from the human ABCD2 gene promoter.  (3222)

Notes: In this paper, proteins were expressed from PCR-generated template DNA using TNT^® Quick for PCR DNA, the TNT^® T7 Wheat Germ Extract System and the E.coli T7 S30 Extract System. (2603)

Notes: The authors used TNT® T7 Quick for PCR DNA to produce protein arrays. Proteins were generated by in vitro translation directly from PCR fragments. (2138)