ABSTRACT
We have demonstrated the isothermal in vitro amplification and multimerization of several different linear DNA targets using only two primers and the strongly strand-displacing exonuclease-negative Bst DNA polymerase. This reaction has been termed linear target isothermal multimerization and amplification (LIMA). LIMA has been compared with cascade rolling-circle amplification and has been found to be less sensitive but to yield similar variable-length multimeric dsDNA molecules. Products from several different LIMA reactions were characterized by restriction analysis and partial sequence determination. They were found to be multimers of subsets of the target sequence and were not purely primer derived. The sensitivities with respect to target concentration of several different LIMA reactions were determined, and they varied from 0.01 amol to 1 fmol. The sensitivity and specificity of LIMA were further tested using E. coli genomic DNA, and the selective amplification of a transposon fragment was demonstrated. A successful strategy for reducing LIMA-dependent background DNA synthesis in rolling-circle amplification embodiments was devised. This entailed the affinity purification of circular DNA templates before amplification.
Subject(s)
DNA Polymerase I/genetics , Geobacillus stearothermophilus/genetics , Nucleic Acid Amplification Techniques/methods , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Bacterial/analysis , DNA, Circular/isolation & purification , Escherichia coli , Geobacillus stearothermophilus/enzymology , Indicators and Reagents , Molecular Sequence Data , Sensitivity and Specificity , StreptavidinABSTRACT
The major open reading frame of banana bunchy top virus (BBTV) DNA-1 encodes a putative replication initiation protein (Rep). In vitro, a fusion protein of BBTV Rep linked to a maltose-binding protein exhibited both site-specific nicking and joining activities. These activities were dependent on the presence of Mg2+ or Mn2+, but did not require ATP. The fusion protein specifically cleaved ssDNA between bases +7 and +8 of a conserved nonanucleotide loop sequence which is present in the virion-strand of the stem-loop common region of each BBTV component. During this reaction, the fusion protein became covalently attached to the 5' end of the 3'cleavage product. After the nicking reactions, the fusion protein was also capable of catalysing the joining of two nicked ssDNA fragments in a site-specific manner. Based on these activities, BBTV Rep would appear to be very similar to the Rep proteins of the geminiviruses.
Subject(s)
Circoviridae/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins , Trans-Activators/metabolism , Viral Proteins/metabolism , Cations, Divalent , Circoviridae/enzymology , Circoviridae/genetics , DNA Helicases/genetics , DNA, Single-Stranded/metabolism , DNA, Viral/metabolism , Fruit/virology , Nucleic Acid Conformation , Oligodeoxyribonucleotides/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/genetics , Viral Proteins/geneticsSubject(s)
Awards and Prizes , Delivery of Health Care, Integrated/standards , Total Quality Management/standards , Consumer Behavior , Data Collection/methods , Delivery of Health Care, Integrated/economics , Delivery of Health Care, Integrated/organization & administration , Economic Competition , Hospital Administrators , Organizational Objectives , Planning Techniques , Self-Assessment , Texas , Total Quality Management/classificationABSTRACT
The rapid, accurate determination of the electrical axis of a patient's heart is very useful for diagnosis. This determination can be made readily by using six electrocardiographic leads. The procedure for interpreting the data from these leads and determining the electrical axis of the heart is charted. This method uses key leads I and aVF to determine the quadrant where the axis is located and secondary leads II, III, aVR, and aVL to find the axial position within the quadrant. When either I or aVF is isoelectric, only one key lead is used.
