Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR.
Nucleic Acids Res
; 47(7): 3619-3630, 2019 04 23.
Article
en En
| MEDLINE
| ID: mdl-30767012
ABSTRACT
Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'â3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bacteriófagos
/
ADN Polimerasa Dirigida por ARN
/
Polimerasa Taq
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos