NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection†

doi:10.7503/cjcu20150427

Abstract

Abstract:

Highly sensitive and selective method for single nucleotide polymorphisms(SNPs) genotyping is of great importance for early diagnosis and treatment of various diseases. By taking rs242557, an Alzheimer’s disease related gene fragment, as a target model, the impact of toehold length on strand displacement reaction was predicted by NUPACK software simulation in order to rationalize the design of duplex DNA probe for high genotyping efficiency. The G-rich DNA sequence was initially blocked in the duplex strand structure. With the addition of target gene fragment to trigger the blocked-domain dehybridization and the toehold-mediated strand displacement reaction, the DNAzyme was formed. Combined with microfluidic chemiluminescence detection, a novel SNP genotyping method had been developed, which was not only label-free, easy to design and exhibited high throughput ability, but also displayed a remarkable specificity to target rs242557 against single base mutation, achieving a discrimination factor of 56 and a detection limit of 1.7 nmol/L with just 2 μL of sample solution consumption.

Key words: NUPACK, Strand displacement reaction, Single nucleotide polymorphisms(SNP), DNAzyme, Microfluidic

CLC Number:

O657.3

TrendMD:

WU Qiwang, SHEN Hong. NUPACK Prediction Assisted of Toehold Induced Strand Displacement Reaction and Its Application in SNPs Genotyping by DNAzyme-catalyzed Microfluidic Chemiluminescence Detection^†[J]. Chem. J. Chinese Universities, 2015, 36(12): 2386.

Figures/Tables 11

Table 1 Sequences of the oligonucleotides used in this work*

Scheme 1 Schematic diagram of sequential injection chemiluminescence system

Scheme 2 Schematic diagram of SNP typing utilizing toehold-mediated strand displacement reaction and DNAzyme-catalyzed chemiluminescence detection

Fig.1 Impact of toehold length on SNP typing (A) Predicted by NUPACK simulations; (B) CL analysis; (C) DF calculation results. The concentrations of P strands, B strands and rs242557 are 150, 300 and 80 nmol/L, respectively.

Fig.2 Native PAGE results(A) and UV-Vis absorption of hemin-oligonucleotide interaction(B) (A) Lane 1: P strand, lane 2: P strand annealed with B strand, lane 3: P/B reacted with rs242557-G, lane 4: P strand annealed with rs242557-G, lane 5: rs242557-G, lane 6: rs242557-A, lane 7: P/B reacted with rs242557-A; (B) hemin: 1.5 μmol/L, P strand: 300 nmol/L, B strand: 600 nmol/L, rs242557 strand: 200 nmol/L. a. Hemin; b. a+P/B; c. b+rs242557-A; d. b+rs242557-G.

Fig.3 Influence of reaction time on DNA detection Control: blank sample solution, sample rs242557-G solution: 80 nmol/L.

Fig.4 Influence of H2O2(A) and luminol(B) concentration on DNA detection Sample: 80 nmol/L rs242557-G solution.

Fig.5 Influence of flow rate on DNA detection Sample: 80 nmol/L rs242557-G solution.

Fig.6 CL response to the different concentrations of sample rs242557-G solution(A) and corresponding calibration plot between net CL response and sample rs242557-G solution from 5 to 400 nmol/L(B) Insert of (B): magnification of the plots in the range of 5—200 nmol/L.

Fig.7 CL responses on control, wild type and mutant type sample solutions Target sample solutions: 80 nmol/L. a. Control; b. rs242557-A; c. rs242557-G; d. rs242557-A34; e. rs242557-G34.

Table 2 DNA detection in biological samples

Sample	Added/(nmol·L^-1)	Found/(nmol·L^-1)	Recovery(%)	RSD(%, n=5)
1	40	36.8	92.0	4.02
2	60	63.4	105.6	2.79
3	80	74.7	93.4	4.51

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