Multi-modal engineering of<i>Bst</i>DNA polymerase for thermostability in ultra-fast LAMP reactions

Paik, Inyup; Pht, Ngo; Shroff, Raghav; Maranhao, Andre C.; Dj, Walker; Bhadra, Sanchita; Ellington, Andrew D.

doi:10.1101/2021.04.15.439918

Cited by 4 publications

(4 citation statements)

References 50 publications

(48 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Over time, several variants of LAMP were introduced, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), multiplex loop-mediated isothermal amplification (M-LAMP), and real-time observation of the product by modifying the original protocol, which allowed for RNA analysis and multiplex detection [12]. As with the RT-PCR variant, RT-LAMP uses reverse transcriptase combined with DNA polymerase to detect RNA sequences or polymerase with reverse transcriptase activity, e.g., Bst 3.0 or OmniAmp [13,14]. The procedure proved to be extremely helpful in diagnosing many RNA viruses [15][16][17].…”

Section: Lamp Methodsmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

Soroкa

Wasowicz

Rymaszewska

2021

Cells

163

100

View full text Add to dashboard Cite

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

show abstract

Section: Lamp Methodsmentioning

confidence: 99%

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

Soroкa

Wasowicz

Rymaszewska

2021

Cells

163

100

View full text Add to dashboard Cite

show abstract

“…The MDCs are summarized in Table and they slightly varied depending on the number of targets. However, the performance of LAMP-HP assay for simultaneous detection of dual or three targets was hindered, potentially due to limitations in raw materials, enzyme activity, and reaction competitions . These factors may have contributed to the challenges faced in achieving optimal performance in multitarget detection.…”

Section: Resultsmentioning

confidence: 99%

Advancing Multiple Detection in RT-LAMP with a Specific Probe Assembled from Plural Three-Way-Junction Structures

Sun,

Zhang,

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

The timely detection of diseases and the accurate identification of pathogens require the development of efficient and reliable diagnostic methods. In this study, we have developed a novel specific multivariate probe termed MRTFP (multivariate real-time fluorescent probe) by assembling strand exchange threeway-junction (3WJ) structures. The 3WJ structures were incorporated into a four-angle probe (FP) and a hexagonal probe (HP), to target the multivariate genes of Salmonella. The FP and HP enable single-step and multiplexed detection in RT-LAMP (real-time loop-mediated isothermal amplification) with exceptional sensitivity and specificity. Encouragingly, real food samples contaminated with Salmonella (Salmonella enteritidis and Salmonella typhimurium) can be readily identified and distinguished with a minimum detectable concentration (MDC) of 10 3 CFU/mL without the need for further culture. The introduction of MRTFP allows for simultaneous detection of dual or three targets in a single tube for LAMP, thereby improving detection efficiency. The MRTFP simplifies the design of robust multivariate probes, exhibits excellent stability, and avoids interference from multiple probe units, offering significant potential for the development of specific probes for efficient and accurate disease detection and pathogen identification.

show abstract

“…Previously, fusion domains such as the DNA-binding domain Sso7d have been used in the construction of thermostable DNA polymerases (Phusion) that have improved properties, such as increased processivity and resistance to PCR inhibitors . In the current instance, we attached a novel fusion domain based on the terminal 47 amino acids of the villin headpiece (HP47) − (Figure ). This headpiece consists of three α-helices that form a highly conserved hydrophobic core and exhibits cotranslational, ultrafast, and autonomous folding properties that may circumvent kinetic traps during protein folding .…”

Section: Resultsmentioning

confidence: 99%

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach

et al. 2021

Self Cite

View full text Add to dashboard Cite

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

show abstract

Multi-modal engineering ofBstDNA polymerase for thermostability in ultra-fast LAMP reactions

Cited by 4 publications

References 50 publications

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

Advancing Multiple Detection in RT-LAMP with a Specific Probe Assembled from Plural Three-Way-Junction Structures

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach

Contact Info

Product

Resources

About