UPDATE blog_clanky SET ctenost = ctenost + 1 WHERE id = 11
Bacteria of Geobacillus species are widely distributed in soil, hot springs, ocean sediment, and are a cause of food deterioration. Bacillus stearothermophilus is a basonym of Geobacillus stearothermophilus, which is a rod-shaped, Gram-positive bacterium, a member of the division Firmicutes. Therefore, the DNA polymerase I (BstI) is an analogue of polymerases from Geobacillus stearothermophilus or other Geobacillus species, such as GstI, GspI, GspISSD. These are moderately thermostable enzymes, a members of polymerase A family, which also belong to high-fidelity polymerases.
As high-fidelity polymerase, BstI enzyme evolved efficient mechanism to avoid incorporation of mismatched dNTPs into polynucleotide chain. The enzyme forms two proofreading complexes. In the first case, the polymerase checks the primer terminus for mismatches while primer terminus is localized between the polymerase and exonuclease subdomains. In the second case, the enzyme adopts a conformation where the primer terminus is bound in the exonuclease subdomain allowing potential excision of the terminal nucleotide residue.
This enzyme does not exhibit any 3’→5’ exonuclease activity potentially caused by deletions located in the 3’ + 5’ exonuclease domains. This feature seems to be common between thermostable polymerases making their structure more compact to be able to tolerate a stringent environmental conditions such as higher temperatures. The catalysis of BstI polymerase is supported by Mg2+, Mn2+, Co2+, and Cd2+ cations.
BstI polymerase possesses a strong strand displacement activity. This is mechanism of rejection of broken 3' single-strand DNA molecule that can form heteroduplex DNA with its complement in an intact duplex DNA. After this, standard base pairing is restored in the original duplex. The previously rejected 3' single-strand DNA molecule anneals with its original complement reforming two intact duplexes. The strand displacement activity represents important feature making BstI polymerase an ideal candidate for isothermal amplification methods (shortly described below).
• Loop-mediated isothermal amplification of DNA (LAMP) – amplification of DNA under isothermal conditions, simple visual detection can be done using fluorescence signal.
• Reverse transcription isothermal multiple-self-matching-initiated amplification (RT-ISMA) – this method resembles RT-LAMP, detection is achieved by combination of reverse transcriptase and DNA polymerase with strand-displacement activity, it is based on generation of multiple self-matching structures (SMSs) by hybrid primers.
• DNA strand displacement amplification (SDA) - method of DNA amplification using combination of a DNA polymerase with strand displacement activity and restriction enzyme.
• Rolling-circle amplification (RCA) – method of amplification of circular DNA template which employs strand displacement mechanism to generate huge amount of circular copies, using fluorescently labelled probes enables easy visual inspection.
• Cross priming amplification (CPA) – other method of isothermal amplification reaction employing continuous primer binding, elongation and strand displacement, it uses multiple primers and probes from which at least one is a cross primer, it is able to amplify specific signal just from four copies of genomic DNA.
• Polymerase chain displacement reaction (PCDR) – method which employs multiple nested primers, it represents a rapid one-tube reaction which can replace normal quantitative PCR (qPCR) assays because of much higher sensitivity.
• Whole genome amplification (WGA) – there are two types: thermocycling and isothermal.
◦ BstI polymerase can by used in isothermal protocol which refers to hyperbranched strand displacement amplification (HSDA), which is also known as multiple-strand misplacement mmplification (MDA), it employs strand displacement activity and random primers causing random initiation points, huge amount of copies of original DNA is generated.
• Sequencing of DNA with high CG content and problematic secondary structures.
• Rapid sequencing from very low amounts of DNA templates.