Helicase-dependent Isothermal DNA Amplification

Overview

Journal EMBO Rep

Specialty Molecular Biology

Date 2004 Jul 13

PMID 15247927

Citations 256

Authors

Myriam Vincent

Yan Xu

Huimin Kong

Affiliations

Soon will be listed here.

Abstract

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

Citing Articles

Swift and portable detection of SC7 through RNA extraction and loop-mediated isothermal amplification using lateral flow device.

Guo S, Zhang Q, Bai N, Yue P, Niu J, Yin C Front Microbiol. 2025; 15():1478218.

PMID: 39831125 PMC: 11739293. DOI: 10.3389/fmicb.2024.1478218.

Enhanced Specificity in Colorimetric LAMP Assay for Detection Using a Combination of Two Additives.

Wong Y, Hau P, Chau E, Ng L, Murillo M, Fung J J Fungi (Basel). 2024; 10(12).

PMID: 39728353 PMC: 11678401. DOI: 10.3390/jof10120857.

Isothermal Detection Methods for Fungal Pathogens in Closed Environment Agriculture.

Cotter A, Dracatos P, Beddoe T, Johnson K J Fungi (Basel). 2024; 10(12).

PMID: 39728347 PMC: 11676205. DOI: 10.3390/jof10120851.

Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting in urine specimens.

Allan-Blitz L, Adams G, Sanders G, Shah P, Ramesh K, Jarolimova J mSphere. 2024; 10(1):e0067724.

PMID: 39688405 PMC: 11774021. DOI: 10.1128/msphere.00677-24.

Point-of-Care Diagnostics Using Self-heating Elements from Smart Food Packaging: Moving Towards Instrument-Free Nucleic Acid-Based Detection.

Hamidizadeh M, Martins R, Bier F Mol Diagn Ther. 2024; 29(1):67-80.

PMID: 39550729 PMC: 11742007. DOI: 10.1007/s40291-024-00753-7.

References

Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G . Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988; 239(4839):487-91. DOI: 10.1126/science.2448875. View

Chong S, Montello G, Zhang A, Cantor E, Liao W, Xu M . Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 1998; 26(22):5109-15. PMC: 147948. DOI: 10.1093/nar/26.22.5109. View

Ali J, Maluf N, Lohman T . An oligomeric form of E. coli UvrD is required for optimal helicase activity. J Mol Biol. 1999; 293(4):815-34. DOI: 10.1006/jmbi.1999.3185. View

Lahue R, Au K, Modrich P . DNA mismatch correction in a defined system. Science. 1989; 245(4914):160-4. DOI: 10.1126/science.2665076. View

Nakai H, Richardson C . The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7. J Biol Chem. 1988; 263(20):9831-9. View

Runyon G, Lohman T . Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA. J Biol Chem. 1989; 264(29):17502-12. View

Fire A, Xu S . Rolling replication of short DNA circles. Proc Natl Acad Sci U S A. 1995; 92(10):4641-5. PMC: 42000. DOI: 10.1073/pnas.92.10.4641. View

Ghedin E, Wang S, Foster J, Slatko B . First sequenced genome of a parasitic nematode. Trends Parasitol. 2004; 20(4):151-3. DOI: 10.1016/j.pt.2004.01.011. View

Bujalowski W, Lohman T . Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects. J Mol Biol. 1989; 207(1):269-88. DOI: 10.1016/0022-2836(89)90455-5. View

10.

Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C . The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997; 25(16):3235-41. PMC: 146890. DOI: 10.1093/nar/25.16.3235. View

11.

Lohman T . Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 1. Irreversible dissociation induced by sodium chloride concentration jumps. Biochemistry. 1984; 23(20):4656-65. DOI: 10.1021/bi00315a022. View

12.

Casas-Finet J, Karpel R . Bacteriophage T4 gene 32 protein: modulation of protein-nucleic acid and protein-protein association by structural domains. Biochemistry. 1993; 32(37):9735-44. DOI: 10.1021/bi00088a028. View

13.

Desplats C, Dez C, Tetart F, Eleaume H, Krisch H . Snapshot of the genome of the pseudo-T-even bacteriophage RB49. J Bacteriol. 2002; 184(10):2789-804. PMC: 135041. DOI: 10.1128/JB.184.10.2789-2804.2002. View

14.

Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gingeras T . Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci U S A. 1990; 87(5):1874-8. PMC: 53586. DOI: 10.1073/pnas.87.5.1874. View

15.

WALKER G, Little M, Nadeau J, Shank D . Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A. 1992; 89(1):392-6. PMC: 48243. DOI: 10.1073/pnas.89.1.392. View

16.

Mechanic L, Frankel B, Matson S . Escherichia coli MutL loads DNA helicase II onto DNA. J Biol Chem. 2000; 275(49):38337-46. DOI: 10.1074/jbc.M006268200. View

17.

Rao U, Williams S, Klei T . Quantification of PCR amplification products of Brugia HhaI repeat DNA using a semiautomated Q-PCR system. Mol Cell Probes. 2002; 16(1):13-23. DOI: 10.1006/mcpr.2001.0393. View

18.

Caruthers J, McKay D . Helicase structure and mechanism. Curr Opin Struct Biol. 2002; 12(1):123-33. DOI: 10.1016/s0959-440x(02)00298-1. View

19.

McReynolds L, DeSimone S, Williams S . Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc Natl Acad Sci U S A. 1986; 83(3):797-801. PMC: 322952. DOI: 10.1073/pnas.83.3.797. View

20.

Andras S, Power J, Cocking E, Davey M . Strategies for signal amplification in nucleic acid detection. Mol Biotechnol. 2001; 19(1):29-44. DOI: 10.1385/MB:19:1:029. View