Summary of Yin-Yang Probes

Matthew Lei, PhD

President and CEO of QuanDx

www.QuanDx.com

QuanDx’ Yin-Yang Probes Patent Status Summary

Target

F Q

+

_

Ex Em

F

_

Q

+

Hybrid: +/Target

+

_

F Q

Ex Em

Yin-Yang Probe(+/- strand)

F: Fluorophore

Q: Quencher

3

4

Real-time qPCR

Digital PCR

As Primers

MicroarrayAnti-sense

FISH Cell

ImagingNGS

F Q

Multifunctional

Real-time qPCR: advantages over TaqMan

5

Easy to Design

High Specificitydetect SNP

MultiplexingSimultaneous

detection of 30 mutations

Cost –effective Synthesis

Yin-Yang Probes

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

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

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.

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.

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.

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 qPCRgenotyping using Yin-Yang Probes

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

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

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

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

Applications by Yin-Yang Probes

• Human in vitro diagnostics

– Human genetics

– Cancer

– Emergency infectious diseases

• Food & water security testing

• Veterinary diagnostics

• BioSurveillance

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 17QuanDx Inc. 17