Supplementary Information
Single-channel multiplexing without melting curve analysis in
real-time PCR
Young-Jo Lee1, Daeyoung Kim1, Kihoon Lee1 & Jong-Yoon Chun1,*
1
Seegene, Inc., Taewon Bldg., 91 Ogeum-ro, Songpa-Gu, Seoul 138-828, South Korea
Supplementary Figure S1. Tagging Oligonucleotide Cleavage and Extension (TOCE) reaction.
(a) TOCE reaction is initiated by hybridization of primers and Pitcher to target sequence. (b) Taq polymerase
with 5’ nuclease activity encounters the target-bound Pitcher, cleaves the Pitcher, and releases the Extender. (c)
The released Extender, complementary to the Capturing portion of the Catcher, anneals to and then is extended.
The resulting Duplex Catcher generates a detectable fluorescence signal. (R: Reporter, Q: Quencher).
c
Supplementary Figure S2. Identification of the targets by detection temperatures.
All the experiments are the same as Figure 2. Experiments were repeated three times and the results were plotted
in one place together. (a) Chlamydia trachomatis (CT) gDNA was used as a target. (b) Neisseria gonorrhoeae
(NG) gDNA was used as a target. (c) Both targets were used as a target.
Supplementary Figure S3. A schematic of how a Ct value of the low Tm target is determined.
The ΔRFU means a change of relative fluorescence unit between two detection temperatures (60°C and 72°C).
The gray bars on the right graphs represent the same arbitrary threshold that was derived from eliminating the
ΔRFU of the high Tm target (CT). The arrows indicate the Ct value of the low Tm target (NG). NTC represents
no target control.
Supplementary Figure S4. Determination of the low Tm target by ΔRFU.
The experiment presented here is the same as Figure 3. It was repeated three times and the data obtained were
shown in one place at the same time. NTC means no target control.
Supplementary Figure S5. Dynamic range of Ct values for the low Tm target and linearity.
(a) Each MuDT reaction (designated as 1 to 5) was performed with different concentration of Neisseria
gonorrhoeae (NG) (low Tm) gDNA (1 ng – 10 fg) without Chlamydia trachomatis (CT) (high Tm) gDNA as
shown in the table. (b) Each MuDT reaction (designated as 6 to 10) was carried out with different concentration
of NG (low Tm) gDNA and 1 ng of CT (high T m) gDNA as shown in the table.
Supplementary Figure S6. Application of a TaqMan probe in MuDT.
MuDT reactions in this experiment were the same as Figure 3 except exploiting a TaqMan probe (CT-P2, 1
pmol) for targeting Chlamydia trachomatis (CT) (high Tm). Two detection temperatures are shown (60°C and
72°C). The first two graphs of each target show the intensity of the unquenched fluorescence signal. ΔRFU
means a change of RFU between two temperatures. Gray bars represent the same arbitrary thresholds that
derived from a line to cut off the ΔRFU of the high Tm target (CT). NTC represents no target control.
Supplementary Table S1. List of oligonucleotide sequences.
Target Description Name Sequence (5’ → 3’)
Neisseria Forward primer NG-F TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG
gonorrhoeae
Reverse primer NG-R CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC
Pitcher NG-P GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG[C3 spacer]
Catcher NG-C [BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(Cal Fluor Red 610)]ACTGCCC
GTATCGCGTAC[C3 spacer]
Chlamydia Forward primer CT-F GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC
trachomatis
Reverse primer CT-R CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA
Pitcher CT-P1 GATTACGCGACCGCATCAGAAGCTGTCATTTTGGCTGCG[C3 spacer]
TaqMan probe CT-P2 [Cal Fluor Red 610]CATCAGAAGCTGTCATTTTGGCTGCG[BHQ-2]
Catcher CT-C [BHQ-2]GCGCTGGATACCCTGGACGA[T(Cal Fluor Red 610)]ATGTGCG
GTCGCGTAATC[C3 spacer]
I: Deoxyinosine
Bold italic letters: Tagging portion of Pitchers
BHQ: Black Hole Quencher
C3 spacer: Three carbon spacer
Underlined letter: Thymine base linked with a fluorescent reporter molecule
Cal Red 610: Cal Fluoro Red 610 Amidite
I: Deoxyinosine
Bold italic letters: Tagging portion of Pitchers
BHQ: Black Hole Quencher
C3 spacer: Three carbon spacer
Underlined letter: Thymine base linked with a fluorescent reporter molecule
Color boxed letter: Mutation site
Cal Red 610: Cal Fluoro Red 610 Amidite
