Summary of Yin Yang Probes
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Yin-Yang probes are double-stranded DNA probes that provide high specificity and fast reaction rates for real-time quantitative PCR (qPCR) and other molecular diagnostic applications. Some key advantages of Yin-Yang probes over other single-stranded probes are their ability to discriminate single nucleotide mismatches, their wider temperature window for discrimination, and their faster hybridization kinetics. Yin-Yang probes have been shown to work well for real-time qPCR, digital PCR, microarrays, fluorescence in situ hybridization, next-generation sequencing, and other applications. They provide an easy to design and cost-effective alternative to probes like TaqMan for multiplex detection of genetic mutations and biomarkers.
Summary of Yin Yang Probes
- 1. Summary of Yin-Yang Probes Matthew Lei, PhD President and CEO of QuanDx www.QuanDx.com
- 2. QuanDx’ Yin-Yang Probes Patent Status Summary
- 3. Target F Q + _ Ex Em F _ Q + Hybrid: +/Target + _ F Q Ex Em Yin-Yang Probe (+/- strand) F: Fluorophore Q: Quencher 3
- 5. Real-time qPCR: advantages over TaqMan 5 Easy to Design High Specificity detect SNP Multiplexing Simultaneous detection of 30 mutations Cost –effective Synthesis Yin-Yang Probes
- 6. Key facts about Yin-Yang Probes • High specificity: – Minus (shorter )strand acts as an additional competitor, which is sufficiently competitive to block nonspecific hybridization but not to interfere with the formation of perfectly matched hybridization. • Fast reaction rate: – Hybridization takes place very fast within 1 min even at 25°C – 10-20 times faster than mismatched targets of hybridization • Wide discrimination temperature window: – There is complete discrimination between perfectly matched target and single nucleotide mismatch targets in the temperature range 30-60°C
- 7. Fast kinetics and high specificity of Yin-Yang Probes Results: • Displacing hybridization takes place very fast within 1 min even at 25°C • Marked reduction in the reaction rate with mismatched oligonucleotide • Yin-Yang probe is listed at the top • Solid curve: perfectly complementary target • Dotted curve: single nucleotide mismatch
- 8. Superior specificity of Yin-Yang Probes Results: • Marked reduction in the reaction rate with mismatched oligonucleotide, suggesting a high degree of discrimination power for Yin-Yang Probes • Open circle: perfectly complementary target(G) • Single nucleotide C (open triangle), A (solid triangle) or T (open square) substitution. • A solution containing no target (solid circle) was used as a control.
- 9. Superior specificity of Yin-Yang Probes When a target oligonucleotide (sequence shown above each panel) was added to the probes mixture, only the double-stranded probe whose probe sequence was perfectly complementary to that target formed a hybrid and emitted its characteristic fluorescent color.
- 10. Comparison to single-stranded probe: high specificity A: Labeling scheme for comparing double-stranded probe with linear/single-stranded probes. A fluorophore was linked to the end of the probe and a quencher was linked to the end of the target. B: While linear probes react equally with matched (solid line) and mismatched (dotted line) targets, double-stranded probes react well with matched (dashed line) but not with mismatched (dot–dash line) targets.
- 11. Unmatched allele discriminating ability of Yin-Yang Probes Real-time qPCR genotyping of ALDH2 ALDH2∗1/2∗1 homozygote, ALDH2∗1/2∗2 heterozygote ALDH2∗2/2∗2 homozygote FAM: solid dots, ROX: empty circles Principle of real-time qPCR genotyping using Yin-Yang Probes
- 12. Comparison to single-stranded Primer: high specificity Using Yin-Yang oligo as PCR primers, not probes, in this application. Upper panel: Yin-Yang (double-stranded) primers where positive template (open circle) and negative template (water, solid circle) Lower panel: conventional single-stranded primer where positive template(open circle) and negative template(water, solid circle) Note: significant non-specific amplification in the conventional single-stranded primer Using SYBR Green I as detection reagents
- 13. In addition as Probes, the advantages using Yin-Yang Primer 1. High specificity as mentioned in previous slide 2. Natural ‘hot-start’ primers, minimizing non-specific annealing in the whole course of amplification, while other ‘hot-start’ methods only function a the beginning of the amplification 3. Color multiplexing ability to enhance in allele-specific amplification
- 14. Comparison to TaqMan • Simple and easy design: Probe design itself is much easier • Cost effective synthesis: 1/5 cost of the current single stranded probe. • High specificity: discriminate single nucleotide mismatch. • High sensitivity: as little as 7.5 pg target DNA could be detected with Yin-Yang probe. • Multiplexing: simultaneous detection of multiple clinically related genes • Spontaneity of reaction: application for biosensors, biochip detection, mRNA tracking
- 15. Comparison to molecular beacon(MB) 1. Simple and easy design: Probe design itself is much easier 2. Lower background and higher sensitivity: • biomolecular duplex (Yin-Yang Probes) denatures faster than an intramolucular duplex (MB) due to the greater entropy change of the Yin-Yang probes. • Sharper transition in the curve of Yin-Yang probes than MB 3. Much wider window between the curves of the perfectly complementary and single mismatch targets with Yin-Yang Probes than with MB, while MB is already know to have a wider window than linear probe like TaqMan
- 16. Applications by Yin-Yang Probes • Human in vitro diagnostics – Human genetics – Cancer – Emergency infectious diseases • Food & water security testing • Veterinary diagnostics • BioSurveillance
- 17. Publications that using Yin-Yang Probes • Li Q, Luan G, Guo Q, Liang J., Nucleic Acids Res. 2002 Jan 15;30(2):E5. • Shengqui W, Xiaohong W, Suhong C, Wei G. Anall Biochem 2002;309:206–211. • Cheng J, Zhang Y, Li Q. Nucleic Acids Res. 2004 Apr 15;32(7):e61. • S. Huang, J. Salituro, N. Tang, K. C. Luk, J. Hackett, P. Swanson,G. Cloherty, W. B. Mak, J. Robinson and K. Abravaya, Nucleic Acids Research, 2007, 35, e101. • R. H. Blair, E. S. Rosenblum, E. D. Dawson, R. D. Kuchta,L. R. Kuck and K. L. Rowlen, Analytical Biochemistry, 2007, 362,213–220. • Wen H, Li Q. J Clin Virol. 2007 Apr;38(4):334-40. Epub 2007 Feb 27. • Ruan L, Pei B, Li Q., Transfusion. 2007 Sep;47(9):1637-42. • Gu Y, Li Q. Clin Biochem. 2007 Nov;40(16-17):1325-7. Epub 2007 Aug 10. • Yang L, Wanqi Liang1, Lingxi Jiang, Wenquan Li, Wei Cao, Zoe A Wilson and Dabing Zhang, BMC Molecular Biology 2008, 9:54 • D. Meserve, Z. Wang, D. D. Zhang and P. K. Wong, Analyst, 2008,133, 1013–1019. • Gidwani V, Riahi R, Zhang D, Wong P, Analyst, 2009, 134, 1675-1681 • Ruan L, Zhao H, Li Q. J Forensic Sci. 2010 Jan;55(1):19-24. Epub 2009 Dec 2. • Li Z, Yang R, Zhao J, Yuan R, Lu Q, Li Q, Tse W. Pediatr Blood Cancer. 2011 Mar;56(3):463-6. • R. Riahi, K. E. Mach, R. Mohan, J. C. Liao and P. K. Wong, Anal. Chem., 2011, 83, 6349–6354 • Zhang D, Chen S, Yin P. Nature Chemistry, 2012, Vol 4, 208-214 • Altan-Bonnet G, Kramer F. Nature Chemistry, 2012, Vol 4, 155-157 • Riahi R, Dean Z, Wu TH, Teitell MA, Chiou PY, Zhang DD, Wong PK. Analyst. 2013 Jun 17 QuanDx Inc. 17