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Development of a Simple Direct and Hot-Start PCR Using Escherichia coli -Expressing Taq DNA Polymerase.
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2023 Jul 13; Vol. 24 (14). Date of Electronic Publication: 2023 Jul 13. - Publication Year :
- 2023
-
Abstract
- Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli ( E. coli ) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli -expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 24
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 37511160
- Full Text :
- https://doi.org/10.3390/ijms241411405