Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection†

doi:10.7503/cjcu20180002

Abstract

Abstract:

Dual enzyme cleavage-based cascade signal amplification for nucleic acids detection was developed. In this system, a modified DNA aligner(MDA) that contains an abasic site was first cleaved by Tth Endonuclease Ⅳ in the presence of target DNA, and then served as an aligner to mediate the cleavage of molecular beacon by nicking endonuclease Nt.BstNBI. These cascade reactions not only overcame the sequence dependence of Nt.BstNBI, but also improved the detection sensitivity. The results showed a good linear correlation between the fluorescence intensity and the logarithm of target DNA concentration(lgc) ranging from 1 pmol/L to 1 nmol/L. Moreover, the proposed method also showed a good capability of identifying single base mutation in target DNA. In addition, this method also features simple probe design and excellent universality. By modifying a small fragment on MDA’s loop, it can be used to sense various target DNAs. Experiments with target DNA spiked in human serum showed the potential of applying this method to real samples.

Key words: Cascade signal amplification, Molecular beacon, Nicking endonuclease, DNA aligner, Nucleic acid detection

CLC Number:

O655.9

TrendMD:

LIAN Xiang, WU Wanghua, FAN Hongliang, ZHANG Yong, ZHANG Tao. Dual Enzyme Cleavage-based Cascade Signal Amplification for Nucleic Acids Detection^†[J]. Chem. J. Chinese Universities, 2018, 39(7): 1427.

Figures/Tables 9

References 24

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Name	Sequence(5'-3')
Molecular Beacon-1(MB-1)	FAM-GCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGC-Dabcyl
Molecular Beacon-2(MB-2)	FAM-CTGCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGCAG-Dabcyl
DNA Aligner(DA)	TCGGTATCTCCTGACTCACGTTTTTCGTGAGTCAACGTCCAGTAC
Modified DNA Aligner(MDA)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCACAT- GAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-FAM (MDA-FAM)	FAM-GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCA- CATGAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-1(MDA-1)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACACCACTCAACCTTCTAGACT- GACTCTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-2(MDA-2)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACTGCTCACATCTGATTCGACACTGT- CACTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Target DNA	ACTCATGTGAGCAAGGTGACACTTG
Target DNA-1(T-1)	GAGTCAGTCTAGAAGGTTGAGTGGT
Target DNA-2(T-2)	GTGACAGTGTCGAATCAGATGTGAGCA
Mutant target DNA-1(MT-1)	ACTCATGTGAGCAAGcTGACACTTG
Mutant target DNA-2(MT-2)	ACTCAcGTGAGCAAGcTGACACTTG
Mutant target DNA-3(MT-3)	ACTgATGTGAGCAAGcTGACACaTG

Name	Sequence(5'-3')
Molecular Beacon-1(MB-1)	FAM-GCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGC-Dabcyl
Molecular Beacon-2(MB-2)	FAM-CTGCAGTGACTTTGTACTGGACGTGGAGATACCGATTTGTCACTGCAG-Dabcyl
DNA Aligner(DA)	TCGGTATCTCCTGACTCACGTTTTTCGTGAGTCAACGTCCAGTAC
Modified DNA Aligner(MDA)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCACAT- GAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-FAM (MDA-FAM)	FAM-GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACCAAGTGTCACCTTGCTCA- CATGAGTTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-1(MDA-1)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACACCACTCAACCTTCTAGACT- GACTCTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Modified DNA Aligner-2(MDA-2)	GCAGTGAACTCAGGTAGAGTCAAC^G^T^C^CAGTACTGCTCACATCTGATTCGACACTGT- CACTCGGTATCTCCTGACTCTACCTGAGTTCACTGC
Target DNA	ACTCATGTGAGCAAGGTGACACTTG
Target DNA-1(T-1)	GAGTCAGTCTAGAAGGTTGAGTGGT
Target DNA-2(T-2)	GTGACAGTGTCGAATCAGATGTGAGCA
Mutant target DNA-1(MT-1)	ACTCATGTGAGCAAGcTGACACTTG
Mutant target DNA-2(MT-2)	ACTCAcGTGAGCAAGcTGACACTTG
Mutant target DNA-3(MT-3)	ACTgATGTGAGCAAGcTGACACaTG

Target sample	1	2	3	4	5	6	7	8	9
Added/(pmol·L^-1)	10.0	10.0	10.0	100.0	100.0	100.0	500.0	500.0	500.0
Found/(pmol·L^-1)	12.1	9.9	8.7	91.3	110.8	95.4	589.0	461.5	522.2
Recovery(%)	117.1	95.1	83.1	90.9	110.4	95.0	117.7	92.2	104.3

Target sample	1	2	3	4	5	6	7	8	9
Added/(pmol·L^-1)	10.0	10.0	10.0	100.0	100.0	100.0	500.0	500.0	500.0
Found/(pmol·L^-1)	12.1	9.9	8.7	91.3	110.8	95.4	589.0	461.5	522.2
Recovery(%)	117.1	95.1	83.1	90.9	110.4	95.0	117.7	92.2	104.3