Alteration of enzymes and their application to nucleic acid amplification (Review)

Abstract

Since the discovery of polymerase chain reaction (PCR) in 1985, several methods have been developed to achieve nucleic acid amplification, and are currently used in various fields including clinical diagnosis and life science research. Thus, a wealth of information has accumulated regarding nucleic acid‑related enzymes. In this review, some nucleic acid‑related enzymes were selected and the recent advances in their modification along with their application to nucleic acid amplification were described. The discussion also focused on optimization of the corresponding reaction conditions. Using newly developed enzymes under well‑optimized reaction conditions, the sensitivity, specificity, and fidelity of nucleic acid tests can be improved successfully.

Figure 1

(A) Domain structures of MMLV RT and AMV RT. (B) A sequence alignment of MMLV RT and AMV RT. A homology search was performed using the search program DNA Data Bank of Japan (DDBJ) CLUSTALW and was revised based on the data of X-ray crystallographic analysis of MMLV RT. Asterisks show homologous amino acid residues. The amino acid residues to be mutated for thermostabilization are marked in bold and are underlined. Leu603 and Glu607 in ref. correspond to Leu593 and Glu597, respectively.

Figure 2

Application of Taguchi's method to optimize the reaction conditions with multiple enzymes. Schematic illustration of the cycle to optimize the reaction conditions is shown.

Figure 3

Model for noise reduction in PCR by helicase. Image of noise reduction in PCR by helicase such as a Tk-EshA is shown in the figure. In the absence of helicase, primers mis-anneal to various homologous regions, resulting in the amplification of noise DNAs. In the presence of helicase shown as a pacman, mis-annealed primers are peeled off and specific primers dominantly anneal to the target region, resulting in the reduced amplification of noise DNAs.

Figure 4

Workflow of the analysis of the error rate in the cDNA synthesis reaction. N₁₄, Key-n, and adaptor α and β indicate the 14-base randomized barcode sequence, five-base key nucleotide sequence, and Ion Proton sequencing adaptor α and β sequences, respectively.

Figure 5

(A) Schematic illustration of the RPA process. (B) Models of SHERLOCK and DETECTR nucleic acid detection systems.