LE probe contains two regions: region I on 3^0 -end hybri-
dizes with adaptor probe and region II on 5^0 -end has com-
plementary sequence with target RNA. A “TTTTT”
sequence is inserted between the two regions.
l A1 5^0 ACA CAGAA AGG GAA ACGAGTCC CGT
TTC CCT TTC.
l A2 5^0 CGT TTC CCT TTCTGTGTGAA AGG GAA
ACGGGA CT.
l X1* 5^0 CGT TTC CCT TTCTGT GTTTTTTATA TCC
CTC GCC GAA TCC TAG ACTCAAAGTAGT CTA
GGA TTC GGC GAG.
X1*^0 consists of two regions: region I on 3^0 -end has the same
sequence with probe X1 and region II on 5^0 -end is a hangout
branch that can initiate the polymerization of hairpin set A.
A “TTTTT” sequence is inserted into the two regions.
l X2 5^0 AGT CTA GGA TTC GGC GAGGGATATCTC
GCC GAA TCC TAG ACTACT TTG.
l Italic sequence indicates the toehold region, bold typeface
indicates loop sequences, and underline indicates stem
sequences.- Target oligo: 5^0 AGT CTA GGA TTC GGC GAGGGATAT
TTTTT CTC TTG GAA AGA AAG TG.
The target oligo can hybridize on its 3^0 -half with the capture
probe on a solid support; the 5^0 -half of “Target oligo” can
initiate the polymerization of hairpin set X*. A “TTTTT”
sequence is inserted between the two regions. - Adaptor: AGT CTA GGA TTC GGC GAGGGA TATTTTTT
ATG CTT TGA CTC AGA AAA CGG TAA CTT C. - 4SYBR Green I: dilute with 4SSC before use, store at 4C
(seeNote 5).
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.3.1 Preparation 1. All hairpin probes are heated to 95C for 2 min and then
allowed to cool to room temperature for 1 h before use.
3.2 Linear HCR by
1.5% Agarose Gel
Electrophoresis
- Mix 7μL of water, 1μL of probe A1, and 1μL of probe A2,
1 μL of target with different concentrations and 1μL of water
for control, incubate at room temperature for 4 h.
192 Yao Xu and Zhi Zheng