Data from 4 additional polymerases (KOD, Paq5000, Herculase II, and Phusion) could not be analyzed because the template was degraded by 3' to 5' exonuclease
activity before acquisition.
The DNA polymerase used in pyrosequencing is the Klenow fragment (bacterial exonuclease
I with the exonuclease
Bir birim Exonuclease
1 ve bir birim SAP PZR urunune eklendi.
In one version of this method, the oligonucleotides are designed to hybridize to the 3' side (“downstream”) of an amplification primer so that the 5'-3' exonuclease
activity of a polymerase digests the 5' end of the probe, cleaving off one of the dyes.
Detection of specific polymerase chain reaction product by utilizing the 5' [right arrow] 3' exonuclease
activity of Thermus aquaticus DNA polymerase.
The DNA polymerase has a 3'[right arrow]5' exonuclease
proofreading activity to maintain high fidelity in the amplified products.
Ontario, Canada) which exhibits 3'[right arrow]5' exonuclease
(proof reading) activity according to manufacturer's instructions.
10 [micro]l of PCR product was digested with Exonuclease
I (10U) and Shrimp Alkaline Phosphatase (1U) for 15 min at 37[degrees]C prior to cycle sequencing to remove leftover primers and dNTPs.
see Figure 1) utilizes the 5' exonuclease
activity of Taq polymerase as originally described in 1991 by Holland et al.
The fluorescence of unbound probe is quenched until the probe binds to the amplicon and the 5' exonuclease
activity of Taq polymerase permanently releases the 5' fluorescent signal.
The probe, which contains both a fluorescence reporter dye at the 5'-end (6-carboxyfluorescein, 6-FAM: maximum emission wavelength = 518 nm) and a quencher dye at the 3'-end (6-carboxytetramethyl rhodamine, TAMRA: maximum emission wavelength = 582 nm), is degraded by the 5'-3' exonuclease
activity of the Taq DNA polymerase, and the resulting fluorescence is detected by a laser in the sequence detector (TaqMan ABI Prism 7700 Sequence Detector System; PerkinElmer).
Also, other mutagenization processes that can be used in combination with, or in lieu of, saturation mutagenesis, including, for example: (a) assembly and/or reassembly of polynucloetide building blocks (including sections of genes &/or of gene families) mediated by a source of exonuclease
activity such as exonuclease
III; and (b) introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.