Incorporation of reporter-labeled nucleotides by DNA polymerases.

Anderson JP, Angerer B, Loeb LA
BioTechniques (2005), Volume 38, Page 257
PubMed entry

Abstract:

The incorporation of fluorescently labeled nucleotides into DNA by DNA ...
The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

Polymerases:

Topics:

Biotech Applications, Nucleotide Analogs / Template Lesions, Nucleotide Incorporation, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) Yes
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
Vent Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) No
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Incorporation of non-standard nucleotides Unspecified Nucleotide analog: Fluorescent dNTPs
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Incorporation of non-standard nucleotides Unspecified Nucleotide analog: Biotin-dNTPs
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Incorporation of non-standard nucleotides Unspecified
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
Taq pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) Yes
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
PGBD Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) Yes
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
Pfu Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) Yes
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. Incorporation of non-standard nucleotides Unspecified
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
T4 Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 3-5' Exonuclease (proofreading) No
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Cloned or native Unspecified
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. 5-3' Exonuclease Unspecified
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Tagged Unspecified
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Full length or truncated Unspecified
Eco Pol I Incorporation of reporter-labeled nucleotides by DNA polymerases. Application name PCR

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