Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction

@article{Sharkey1994AntibodiesAT,
  title={Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction},
  author={David J. Sharkey and Edward R. Scalice and Kenneth George Christy and Susan Melissa Atwood and John L. Daiss},
  journal={Bio/Technology},
  year={1994},
  volume={12},
  pages={506-509}
}
We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20–40°C) and release fully active TaqPol… 

Approach to Reaction Using Taq

The results of these experiments suggest that TaqStart antibody inhibits nonspecific products and primer-dimers formation by blocking Taq DNA polymerase activity until the reaction components are heated in the thermal cycler (hot start).

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors, which makes the enzyme ideally suited for cutting-edge PCR-applications.

Polymerase Chain Reaction (PCR) in Chemical Biology

PCR applications include cloning of genomic DNA, preparing DNA for sequencing, site-directed mutagenesis and recombination, genetic fingerprinting for forensic purposes, detection and identification of infectious agents, prenatal diagnosis of genetic diseases, identification of allelic sequence variations, and gene-expression analysis.

Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.

It is shown that the novel cold-sensitive mutants of Klentaq (an N-terminal deletion variant) DNA polymerase can provide a hot start for PCR and exhibit slightly improved fidelity.

Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities.

It is found that nonspecific background amplification includes an early phase and a late phase, which limits the sensitivity of EXPAR, and observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis.

Magnesium precipitate hot start method for PCR.

It is demonstrated that the method is as effective as a manual hot start (addition of magnesium at or above primers'annealing temperatures) for several target gene amplifications which require a hot start.
...

References

SHOWING 1-10 OF 11 REFERENCES

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

Improved amplification performance is evident for target copy numbers below approximately 10(3), and when mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction.

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR

Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro.

This multiphasic approach enabled us to detect specific human T cell leukemia virus type I-homologous regions in several HTLV-I-seronegative patients with T cell lymphoma, as well as variants of HT LV-I and human immunodeficiency virus type 1 in patients with prototype disease.

Recent advances in the polymerase chain reaction

Progresses ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences and some recent developments in instrumentation, methodology, and applications of the PCR are presented.

DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells.

This method of DNA amplification made it possible to obtain results within 3 days, whereas virus isolation takes up to 3 to 4 weeks, and may therefore be used to complement or replace virus isolation as a routine means of determining HIV-1 infection.

Increased PCR sensitivity by using paraffin wax as a reaction mix overlay.

The use of inexpensive paraffin wax as a reaction mix overlay eliminated problems associated with the presence of mineral oil while being more practical and safer in handling potentially contaminated clinical samples.

The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion.

  • K. Mullis
  • Medicine
    PCR methods and applications
  • 1991
This research presents a meta-anatomy of the immune checkpoint disease and its role in cancer diagnosis and treatment, and some of the mechanisms behind its development have been described.

Polymerase chain reaction strategy.

The aim of this work is to contribute towards the humanizing of PCR and its application in the fields of medicine and public health.