Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications

Abstract

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.

Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance

“…The Gss-pol nucleotide sequence was amplified using Gss-F1 and Gss-R1 primers with NdeI and NotI restriction sites, allowing the in-frame ligation into the pET23a vector (Novagen, USA). PCR was carried out using previously constructed pQE-LF-Gss ( 15 ), which contains the nucleotide sequence of the large fragment of DNA polymerase I from Geobacillus sp. 777 .…”

“…Previously, we cloned and characterized the Bst- like polymerase Gss -polymerase, DNA polymerase I from Geobacillus sp. 777 suitable for isothermal amplification of DNA ( 15 ). In the present work, we designed a set of chimeric polymerases on the basis of Bst -like Gss -polymerase and DNA-binding domains using the DNA-binding domain of DNA ligase Pyrococcus abyssi ( 16 ) and Sto7d protein, a counterpart of Sso7d from Sulfolobus tokodaii .…”

Thermal Analysis of a Disposable, Instrument-Free DNA Amplification Lab-on-a-Chip Platform

“…In this paper, loop-mediated isothermal amplification (LAMP) is the protocol of choice, which provides the maximum yield within 60–65 °C temperature [ 7 ]. In our LAMP assay (detailed in our previous works [ 35 , 36 , 37 ]), we use Bsm polymerase, whose activity is higher than 80% within the 57–62 °C reaction temperature range, as demonstrated by Oscorbin et al [ 38 ]. Therefore, we define this temperature range as the target for our heating system.…”

Выявление мутации Ser450Leu в гене rpoB Mycobacterium tuberculosis методом аллель-специфичной изотермической петлевой амплификации ДНК

“…Gss-полимеразы из Geobacillus sp. 777 [15]. В случае проведения LAMP в режиме реального времени добавляли интеркалирующий краситель SYTO-9 до концентрации 2 мкМ [16] Нами были также предприняты попытки улучшения индекса дискриминации мутации с помощью изменения концентрации бетаина, а также путем добавления диметилсульфоксида (ДМСО), который обладает способностью увеличивать специфичность стандартной ПЦР [19].…”