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.

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

4. 4
Real-time
qPCR
Digital
PCR
As
Primers
Microarray
Anti-
sense
FISH
Cell
Imaging
NGS
F Q
Multifunctional

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
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