Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.
BMC Biotechnol
; 24(1): 17, 2024 Apr 02.
Article
em En
| MEDLINE
| ID: mdl-38566117
ABSTRACT
Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 â 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Uracila
/
DNA Polimerase Dirigida por DNA
Idioma:
En
Revista:
BMC Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Dinamarca