Strand displacement assays

C trachomatis and CT, Chlamydia trachomatis N, gomrrhoeae and GC, Neisseria gonorrhoeae PCR, polymerase chain reaction SDA, strand displacement assay TMA, transcription-mediated amplification. 

Branch capture reactions (strand displacement assays)... 

Fig. 8.7, In strand-displacement assays, two regions are distinguished in an analyte (S and T ). The T reacts with the T (target C) and displaces a short signal sequence from this complex by branch migration (D). The liberated signal sequence (S ) is removed from the well (E) and detected (F).

Fig. 3. Strand displacement assay. Polymerization reactions were performed at 68° as described in the text using f Turbo (lanes 3,4), lyu (lane 5), or Taq (lane 6) DNA polymerase. (Stratagene s PfuTurbo DNA polymerase is a blend of cloned Pfu DNA polymerase and a proprietary PCR enhancing factor.) The blocking oligo was added to reactions shown in lanes 4-6. Lane 1 shows the radiolabeled 20 bp size marker (two bands are resolved on the denaturing gel for each size marker), while lane 2 is a no enzyme (template only) control.

HCV and HIV-1). The bDNA assay is being much employed for the quantification of messenger RNA. Moreover, for the detection of viral and pathogenic disorders based on alkahne-phosphatase-sensitive dioxetanes, several assay methods are available these include the Polymerase-Chain-Reaction (PCR) amphfication, probe ligation, strand-displacement amplification and the ligase chain reaction. ... 

Recently, a new in vivo assay of cytoskeletal organization was introduced into plant cell biology, in which a laser trap can be used to displace various cytoplasmic structures [103,104]. The power required to displace transvacuolar strands, which consist of MTs and MFs, can be used to assay cytoskeletal tension. This is the so-called cell optical displacement assay (CODA). CODA experiments have been performed to estimate the... 

In addition to PCR, there are many other technologies to amplify nucleic acids. For example, ligation-based amplification or ligase chain reaction uses sequence-directed oligonucleotide primers and thermostable DNA ligase to assay point mutations, deletions, or insertions in DNA. Strand-displacement amplification uses the inherent strand-displacement activity of DNA polymerases to conduct DNA amplification at a constant temperature. Transcription-based methods such as nucleic acid sequence-based amplification (NASBA) involve in vitro RNA transcription. NASBA and most other transcription-based... 

Detection of the unwound product (ssNA) in the high-throughput helicase assay is dependent on time, substrate, and enzyme concentration, and is prevented by the presence of an inhibitor. The assay format can utilize double-stranded DNA as well as RNA substrates, making the assay amenable to use with both DNA and RNA helicases. Traditional RNA or DNA helicase assays require separation of radiolabeled double-stranded substrate from unwound product by polyacrylamide gel electrophoresis (PAGE). Quantitation of a conventional helicase gel assay requires the use of a phosphorimager, or dissection of the radiolabeled separated substrate and displaced products from the dried gel and detection of associated cpm with a scintillation counter. 

Table V shows that some activity is observed for metal salts in lethality studies. These metal ions are not as active as the inert complexes in the lethality assay, and none of these ions has been found to be active in reversion studies. Perhaps the difference is that the metal ions rapidly come to equilibrium in the cell and form kinetically labile and non-toxic complexes with DNA. As strand separation occurs, the labile ions that are on the DNA can be rapidly displaced and thus are not able to interfere with replication. On the other hand, the inert ions undergo substitution at rates which are slow compared to cell division. When they undergo ligand substitutions and become attached to DNA, they remain fixed to the DNA long enough to cause errors during replication.

The DNA-binding domain of mPARP-2 (aa 1-65 Fig. 2) was identified on the basis of its capacity to bind damped DNA in a Southwestern assay. Using precisely defined DNA ends, we demonstrated that purified mPARP-2 binds specifically to a gap of one nucleotide (F. 3A) and protects about 10 nucleotides. In contrast to PARP-1, PARP-2 does not bind to a break (Fig. 3B). The affinity of PARP-2 for the inside of the double helix can also be visualized by electron microscopy (Fig. 3C) where the protein accumulates and seems to displace one of the two DNA strands (arrows) when a break is present. 

One assay we use to monitor exonuclease activity was adapted from a published method that employs uniformly labeled DNA. Using double-stranded labeled DNA templates, we can determine specificity by measuring whether cpms increase (5 - -3 exonuclease) or decrease (3 ->5 exonuclease) upon the addition of dNTPs (10-100 pJl ). For specifically monitoring DNA polymerases with 5 3 structure-specific endonuclease activity, duplex DNA templates which contain displaced 5 ends are preferred. As is the case for polymerase activity measurements, exonuclease assays are significantly influenced by reaction conditions, and salt concentration, incubation temperature, and DNA concentration should be specifically optimized for each DNA polymerase. 

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