Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review

Abstract

Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.

Thermostable DNA ligases from hyperthermophiles in biotechnology

“…Besides LCR, ligase detection reaction (LDR), ligation amplification reaction (LAR), and gap-LCR have also been developed with thermostable DNA ligases ( Wu and Wallace, 1989 ; Abravaya et al, 1995 ; Li et al, 2022 ). Additionally, thermostable DNA ligases can also be used for rolling-circle amplification (RCA) in the presence of padlock probes, which is an alternative method for detecting SNP ( Yan et al, 2023 ).…”

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification

“…The detection of single nucleotide variants (SNVs), commonly known as point mutations, plays a pivotal role in advancing our understanding of genetic diseases, cancer development, evolutionary biology, and personalized medicine. 1 Ligasebased amplification and detection techniques have been extensively utilized for SNVs discrimination in pathogen detection 2 genetic diseases 2,3 and cancer diagnosis. [4][5][6][7] In these techniques, a DNA ligase is employed to favor the amplification of one DNA target versus the target which carries a mismatch at the ligation site.…”

“…[4][5][6][7] In these techniques, a DNA ligase is employed to favor the amplification of one DNA target versus the target which carries a mismatch at the ligation site. 1 Ligation-based amplification techniques can be roughly divided into two main categories: those that use a ligase for the mismatch discrimination and a polymerase with strong strand displacement activity for the amplification of the "ligated" target like the Rolling Circle Amplification (RCA), the Strand Displacement Amplification (SDA) 2 and the past few years the ligase-based isothermally exponential amplification (LIEXA) 8 and Loop-mediated isothermal amplification (LAMP) 9 reactions. The other category includes methods mainly based on the ligation for the detection such as the Oligonucleotide Detection Assay (OLA) and the Ligase Detection Reaction (LDR), the Ligase Chain Reaction (LCR) and LCR variants (Gap-LCR, Nested-LCR).…”

Microwave-Dependent Thermo-Release Approach for Oligonucleotides 5′-Phosphorylation

“…2 Due to their biological significance, phosphorylated oligonucleotides find extensive applications in various molecular techniques, 3 serving as linkers and adapters 4 in cloning and gene regulation, 5 as well as in the ligase chain reaction. 6 Nevertheless, in the routine chemical synthesis of nucleic acids, the synthetic oligonucleotide ends lack phosphorylation. Introducing the phosphate moiety requires an additional step using a dedicated reagent.…”

“…The incorporation of phosphate into nucleotides or at the 5′-end of a polynucleotide is one of the most widely utilized techniques in the solid-phase chemical synthesis of oligonucleotides . Due to their biological significance, phosphorylated oligonucleotides find extensive applications in various molecular techniques, serving as linkers and adapters in cloning and gene regulation, as well as in the ligase chain reaction . Nevertheless, in the routine chemical synthesis of nucleic acids, the synthetic oligonucleotide ends lack phosphorylation.…”