Incorporation of reporter-labeled nucleotides by DNA polymerases.

BioTechniques (2005), Volume 38, Page 257

Abstract:

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 Anderson JP2005 3-5' Exonuclease (proofreading) Yes
Vent Anderson JP2005 Cloned or native Unspecified
Vent Anderson JP2005 5-3' Exonuclease Unspecified
Vent Anderson JP2005 Tagged Unspecified
Vent Anderson JP2005 Full length or truncated Unspecified
Vent Anderson JP2005 Application name PCR
Taq pol I Anderson JP2005 3-5' Exonuclease (proofreading) No
Taq pol I Anderson JP2005 Incorporation of non-standard nucleotides Unspecified Nucleotide analog: Fluorescent dNTPs
Taq pol I Anderson JP2005 Incorporation of non-standard nucleotides Unspecified Nucleotide analog: Biotin-dNTPs
Taq pol I Anderson JP2005 Incorporation of non-standard nucleotides Unspecified
Taq pol I Anderson JP2005 Cloned or native Unspecified
Taq pol I Anderson JP2005 5-3' Exonuclease Unspecified
Taq pol I Anderson JP2005 Tagged Unspecified
Taq pol I Anderson JP2005 Full length or truncated Unspecified
Taq pol I Anderson JP2005 Application name PCR
PGBD Pol I Anderson JP2005 3-5' Exonuclease (proofreading) Yes
PGBD Pol I Anderson JP2005 Cloned or native Unspecified
PGBD Pol I Anderson JP2005 5-3' Exonuclease Unspecified
PGBD Pol I Anderson JP2005 Tagged Unspecified
PGBD Pol I Anderson JP2005 Full length or truncated Unspecified
PGBD Pol I Anderson JP2005 Application name PCR
Pfu Anderson JP2005 3-5' Exonuclease (proofreading) Yes
Pfu Anderson JP2005 Cloned or native Unspecified
Pfu Anderson JP2005 5-3' Exonuclease Unspecified
Pfu Anderson JP2005 Tagged Unspecified
Pfu Anderson JP2005 Full length or truncated Unspecified
Pfu Anderson JP2005 Application name PCR
T4 Anderson JP2005 3-5' Exonuclease (proofreading) Yes
T4 Anderson JP2005 Incorporation of non-standard nucleotides Unspecified
T4 Anderson JP2005 Cloned or native Unspecified
T4 Anderson JP2005 5-3' Exonuclease Unspecified
T4 Anderson JP2005 Tagged Unspecified
T4 Anderson JP2005 Full length or truncated Unspecified
T4 Anderson JP2005 Application name PCR
Eco Pol I Anderson JP2005 3-5' Exonuclease (proofreading) No
Eco Pol I Anderson JP2005 Cloned or native Unspecified
Eco Pol I Anderson JP2005 5-3' Exonuclease Unspecified
Eco Pol I Anderson JP2005 Tagged Unspecified
Eco Pol I Anderson JP2005 Full length or truncated Unspecified
Eco Pol I Anderson JP2005 Application name PCR

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