Notes: RT-PCR and PTT detected truncated peptides produced from the mRNA transcripts of mutant CFTR genes. Due to the large size of the gene for CFTR, RT-PCR and the PTT are attractive tests for screening for frameshift mutations in the gene. The PTT was performed using an aliquot of nested PCR product (consisting of the CFTR cDNA fused to the T7-promoter) in the TNT® T7 Coupled Reticulocyte Lysate System. (1869)

Notes: The fmol^® DNA Cycle Sequencing System was used to determined the sequence of cloned PCR products. (1140)

Notes: Taq DNA Polymerase was used in this study. (2013)

Notes: The gene for 20.5K protein of Ad3/7 was amplified by PCR and used as a template for the TNT® Coupled Reticulocyte Lysate System using [35S]cysteine. The proteins were immunoprecipitated with antisera to a 20.5K-specific synthetic peptide. (1920)

Notes: [³⁵S]methionine-labeled ApCREB2 and ApC/EBP were translated in the TNT^® Coupled Reticulocyte Lysate System. In vitro interaction assays were performed with the labeled ApCREB2 and ApC/EBP and with GST fusions of ApCREB2 and its deletion mutants. ApCREB2 forms weak homodimers, and ApCREB2 interacts with ApC/EBP. (1781)

Notes: The Altered Sites^® II in vitro Mutagenesis System was used to produce 14 consecutive 4bp deletions in a putative response element. (1157)

Notes: RBP-Jk protein was transcribed and translated in vitro using the TNT^® Coupled Reticulocyte Lysate System. The protein was labeled with [³⁵S] and used in mobility shifts with PCR-generated probes to demonstrate the binding of the RBP-Jk protein to the RBP-Jk site in the Cp promoter. (1800)

Notes: Promega's Taq DNA Polymerase was used in this study. (1957)

Notes: This report details the synthesis, characterization and application of a photocleavable biotin derivative (PCB) for use in detection of biomolecules, without the limitations of irreversibility (also see patent, below). The reagent, PCR-NHS contains a biotin moiety linked through a spacer arm to a photocleavable moiety that reacts selectively with amino groups on a target molecule to form a stable carbamate. The conjugate undergoes a photoreaction at 300nm, with rapid release of the unmodified target molecule. (1944)

Notes: RT-PCR and PTT were used to screen for mutations associated with mental retardation in DMD patients in the 3' coding region of the dystrophin gene. The TNT^® T7 Coupled Reticulocyte Lysate System was used for in vitro transcription/translation reactions. (0244)

Notes: RT-PCR and the protein truncation test (PTT) were used to detect BRCA1 mutations. RT-PCR products were used as templates for protein synthesis in the TNT^® T7 Coupled Reticulocyte Lysate System. The products were analyzed by SDS-PAGE. Eight predisposing mutations were identified. (1053)

Notes: The protein truncation test (PTT) was used to monitor the integrity of the dystrophin ORF in DMD patients. The TNT® T7 Coupled Reticulocyte Lysate System was used for PTT analysis in the presence of [35S]methionine. (In ~66% of DMD patients, a deletion in dystrophin is the causative agent.) Discontinuous SDS-PAGE is used to separate the proteins produced, and individuals carrying certain mutations (e.g., nonsense) are easily detected. (1870)

Notes: Four troponin T cDNAs were transcribed and translated with the TNT® Coupled Reticulocyte Lysate System. Two of the four proteins were immunoprecipitated with polyclonal antisera to a 10-residue sequence that is encoded by an alternatively spliced exon. The results show that there is alternative splicing of two exons in the amino-terminal half of troponin T. (1890)

Notes: A 2kb fragment of the APC exon 15 (a mutation cluster region) was generated by PCR amplification from patient and tumor DNA. The PCR products were transcribed and translated in the TNT^® T7 Coupled Reticulocyte Lysate System and subjected to the protein truncation test (PTT), the PTT revealed that several truncation mutations in exon 15 were responsible for different familial adenomatous polyposis cases. (0216)

Notes: The inhibitor was used for protection of cells and colonies following RT-PCR. (1672)

Notes: The CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) was used to measure cell proliferation. (1732)

Notes: RelA and NFKappaB1, tagged with HA, were synthesized from PCR products using the TNT® T7 Coupled Reticulocyte Lysate System. The proteins were coimmunoprecipitated with anti-HA and analyzed by SDS-PAGE. Multimerization was demonstrated for the proteins with the formation of the heterodimer being stronger than the homodimers. The proteins were also tested in gel mobility shift assays with kappaB DNA probes to demonstrate DNA binding specificity. (1830)

Notes: Enzymatically active diphtheria toxin was formed in vitro by transcription/translation from PCR products carrying the A- and B-fragments of the toxin. T3 RNA Polymerase was used for in vitro transcription and the Rabbit Reticulocyte Lysate System was used for translation of proteins. Both wild type and mutant proteins were produced and assayed. The synthesized proteins were tested for trypsin sensitivity, ability to inhibit protein synthesis, binding and translocation in Vero cells. Considerable alterations can be made to the C-terminal region of the protein without blocking translocation. Some mutant toxins were less toxic than wild type toxins in unnicked form, but when nicked most exhibited the same toxicity. (2226)

Notes: RNA isolated from peripheral blood lymphocytes of DMD patients was used as a template for RT-PCR of a region of the dystrophin gene. The protein truncation test was performed using proteins transcribed and translated from the RT-PCR products with the TNT^® T7 Coupled Reticulocyte Lysate System. The translation products were analyzed by SDS-PAGE. PTT detects nonsense but not missense mutations. (0492)

Notes: PTT was performed on the RNA of a DMD patient and his mother, a carrier of the DMD mutation. Total RNA was isolated from blood. RT-PCR and 'boosted' PCR was used to amplify a partial cDNA of the gene with a T7 promoter. The engineered template was expressed in vitro with the TNT^® T7 Coupled Reticulocyte Lysate System in the presence of [³H]leucine followed by SDS-PAGE. The PTT may be used to screen the entire coding region of the DMD gene in 5-10 reactions using 5 overlapping primer sets for the primary PCR and 10 nested primer sets for the 'boosted' PCR. (0493)

Notes: The Wizard^® DNA Clean Up System was used to clean up DNA for methylation specific PCR. (0333)