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Vol. 36, No. 3 (2004) BioTechniques 1
BioTechniques
®
The Journal of Laboratory Technology for Bioresearch
Volume 36, No. 3 March, 2004
Annealing control primer system for
identification of differentially expressed
genes on agarose gels
Yun-Jee Kim, Chae-Il Kwak, Young-Yun Gu, In-Taek Hwang,
and Jong-Yoon Chun
Seegene Life Science Laboratory, Seoul, South Korea
BioTechniques 36:424-434 (March 2004)
We developed GeneFishing technology, an improved method for the identification of differen-
tially expressed genes (DEGs) using our novel annealing control primer (ACP) system. Because
of high annealing specificity during PCR using the ACP system, the application of the ACP to
DEG discovery generates reproducible, authentic, and long (100 bp to 2 kb) PCR products that
are detectable on agarose gels. To demonstrate this method for gene expression profiling, Gene-
Fishing technology was used to detect genes that are differentially expressed during develop-
ment using total RNAs isolated from mouse conceptus tissues at 4.5–18.5 days of gestation. Ten
DEGs (DEG1–10) were isolated and confirmed by Northern blot hybridization. The sequence
analysis of these DEGs showed that DEG6 and DEG10 are unknown genes.
INTRODUCTION
Techniques designed to identify dif-
ferentially expressed genes (DEGs) in
cells under various physiological stages
or experimental conditions (e.g., during
developmental or neoplastic differen-
tiation or pharmacological treatment)
have become pivotal in modern biol-
ogy. The difficulty of identifying (or
“fishing out”) a gene responsible for
a specialized function during a certain
biological stage often arises because
the gene is expressed at low levels,
whereas the bulk of mRNA within a
cell are highly abundant transcripts (1).
To screen for DEG transcripts in low
concentrations, PCR amplification is
required. One screening method, dif-
ferential display, requires PCR using
short arbitrary primers and is described
by Liang and Pardee (2). Although
this method is simple, rapid, and only
requires small amounts of total RNA,
many investigators have experienced
significantly high false-positive rates
(1,3) and poor reproducibility of results
(4) because of nonspecific annealing by
short arbitrary primers. The use of short
primers (10–13 bp) usually requires
low annealing temperatures about
40°–45°C for all PCR cycles; these low
temperatures cause nonspecific primer
annealing. The use of longer primers
(18–20 bp) make it possible to increase
the annealing temperature to 60°C af-
ter 1–4 initial cycles at 40°–45°C (5,6).
However, the additional tail sequences
of longer primers are involved in non-
specific annealing to the cDNA tem-
plate during PCR cycles at low anneal-
ing temperatures (7).
We recently developed the annealing
control primer (ACP) system (7) that
uses primers that anneal specifically
to the template and allows only genu-
ine products to be amplified, a process
that eliminates false-positive results.
The ACP system is based on two prin-
ciples: the unique tripartite structure of
the primers, which have distinct 3′- and
5′-end regions that are separated by a
polydeoxyinosine [poly(dI)] linker, and
the interaction of each region during
two-stage PCR. We adapted the ACP
system for the identification of DEGs
involved in mouse development.
MATERIALS AND METHODS
First-Strand cDNA Synthesis
Total RNAs extracted from mouse
conceptus tissues at different devel-
opmental stages [4.5, 11.5, and 18.5
days postcoitus (dpc)] were used for
the synthesis of first-strand cDNAs by
reverse transcriptase, as described by
Hwang et al. (7). Reverse transcrip-
tion was performed for 1.5 h at 42°C
in a final reaction volume of 20 µL
containing 3 µg of the purified total
RNA, 4 µL of 5× reaction buffer (Pro-
mega, Madison, WI, USA), 5 µL of
dNTP (each 2 mM), 2 µL of 10 µM
cDNA synthesis primer, oligo(dT)15,
oligo(dT)15 tail, or oligo(dT)15 ACP
(Table 1), 0.5 µL of RNasin® RNase
Inhibitor (40 U/µL; Promega), and 1
µL of Moloney murine leukemia vi-
rus reverse transcriptase (200 U/µL;
Promega). First-strand cDNAs were
diluted by the addition of 80 µL of ul-
tra-purified water.
ACP-Based GeneFishing PCR
Second-strand cDNA synthesis and
subsequent PCR amplification were
conducted in a single tube. Second-
strand cDNA synthesis was conducted
at 50°C (low stringency) during one cy-
cle of first-stage PCR in a final reaction
2 BioTechniques Vol. 36, No. 3 (2004)
SHORT TECHNICAL REPORTS
volume of 49.5 µL containing 3–5 µL
(about 50 ng) of the diluted first-strand
cDNA, 5 µL of 10× PCR buffer plus Mg
(Roche Applied Science, Mannheim,
Germany), 5 µL of dNTP (each 2 mM),
1 µL of oligo(dT)15, oligo(dT)15 tail,
or oligo(dT)15 ACP (10 µM), and 1
µL of 10 µM arbitrary primer (Table
1, 10-mer, 10-mer tail, or 10-mer ACP)
preheated to 94°C. The tube containing
the reaction mixture was held at 94°C
while 0.5 µL of Taq DNA Polymerase
(5 U/µL; Roche Applied Science) was
added to the reaction mixture. The PCR
protocol for second-strand synthesis
was one cycle at 94°C for 1 min, fol-
lowed by 50°C for 3 min, and 72°C for
1 min. After second-strand DNA syn-
thesis was completed, the second-stage
PCR amplification protocol was 40 cy-
cles of 94°C for 40 s, followed by 65°C
for 40 s, 72°C for 40 s, followed by a
5-min final extension at 72°C. The am-
plified PCR products were separated in
2% agarose gel stained with ethidium
bromide. The differentially expressed
bands were extracted from the gel by
using a GENECLEAN® II Kit (Qbio-
gene, Carlsbad, CA, USA), directly
cloned into the pGEM®-T Easy vec-
tor (Promega) without reamplification
of the recovered bands, and sequenced
with ABI PRISM® 310 Genetic Analyzer
(Perkin Elmer, Boston, MA, USA).
Northern Blot Analysis
Northern blots of full-stage mouse
conceptus tissue (4.5–18.5 dpc) and of
multiple adult mouse tissues (Seegene,
Seoul, South Korea), each lane contain-
ing 20 µg of total RNA, were hybrid-
ized overnight with the 32P-labeled
cDNA probe in QuikHyb® solution
(Stratagene, La Jolla, CA, USA) as pre-
viously described (8). The probe cDNA
fragments for each DEG were pre-
pared by PCR using their correspond-
ing clones as templates. The fragments
were amplified by using the universal
(tail) sequences of oligo(dT)15 ACP
and arbitrary ACPs JYC5 (5′-CTGT-
GAATGCTGCGACTACGAT-3′) and
JYC4 (5′-GTCTACCAGGCATTCGC-
TTCAT-3′).
RESULTS AND DISCUSSION
Principle of GeneFishing Technology
To apply the ACP primer system
to differential display, first-strand
cDNAs are synthesized by reverse
transcription using oligo(dT)15 ACP
as a primer. This method requires only
a single cDNA synthesis for each dif-
ferent RNA sample, in contrast to the
multiple cDNA reactions required for
differential display methods (2,9). Us-
ing first-strand cDNAs as templates,
second-strand cDNAs are synthesized
during one cycle of first-stage PCR us-
ing an arbitrary ACP primer and an ini-
tial annealing temperature (50°–53°C).
In other protocols, the presence of re-
sidual oligo(dT) primers used in first-
strand cDNA synthesis results in high
background noise because oligo(dT)
can potentially anneal to all cDNAs in
the reaction mixture. In our approach,
oligo(dT)15 ACP primer and arbitrary
ACP coexist in the same reaction tube,
but the 3′-end core region [(dT)15] of
oligo(dT)15 ACP cannot anneal to the
first-strand cDNAs at the initial an-
nealing temperature due to the lower
annealing temperature required. How-
ever, such annealing temperature does
permit the 3′-end core sequence (10-
mer) of the arbitrary ACP to anneal to a
specific template site. This is one of the
unique features of GeneFishing tech-
nology; because of the selective hybrid-
izing ability of the ACP system during
first-stage PCR, background from re-
sidual oligo(dT) primers can be elimi-
nated. Second-strand cDNAs are then
amplified during second-stage PCR at
a second annealing temperature (65°C),
which are high-stringency conditions,
using the sequences at the 3′ and 5′ ends
of the second-strand cDNAs as the tem-
plates for the amplification priming se-
quences. During the second-stage PCR,
the 3′-end core region sequences alone
of the oligo(dT)15 ACP or the arbitrary
ACP primer cannot anneal to the cDNA
templates in such high-stringency con-
ditions, another selective hybridization
feature of GeneFishing technology.
Consequently, second-strand cDNAs
can be amplified at almost the theo-
retical optimum of a 2-fold increase in
product for each cycle of second-stage
PCR amplification.
To examine the above features of
the ACP system using differential dis-
play, we compared ACPs with the con-
ventional short or longer primers. The
conventional longer primers have the
same structure of the ACP except for
the poly(dI) linker. The conventional
short oligo(dT)1 5 and arbitrary (10-
mer) primers were not stable enough
to generate any products under condi-
tions such as the above (Figure 1, lanes
1–3). The use of the conventional lon-
ger oligo(dT) primer was confounded
with high background (Figure 1, lane
4). Further, the results with the con-
ventional longer primers showed a
quite different pattern from the results
generated with ACP primers (Figure 1,
lanes 6 and 9). According to data ob-
tained from the cloning and sequenc-
ing of the amplified products, the
universal (tail) sequence of the con-
ventional longer primers was involved
in primer annealing in an unpredicted
manner (data not shown). Our results
are consistent with the results from the
amplification of a target nucleotide
sequence previously described (7), in
which nontarget universal sequences
of the longer conventional prim-
ers are involved in primer annealing,
Table 1. Primer Sequences Used in cDNA Synthesis and ACP-Based GeneFishing PCR
Use Primer Sequence
cDNA
Synthesis
Primer
Oligo(dT)15
Oligo(dT)15 tail
Oligo(dT)15
ACP
5′-TTTTTTTTTTTTTTT-3′
5′-CTGTGAATGCTGCGACTACGATTTTTTTTTTTTTTTT-3′
5′-CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTT-
3′
Arbitrary
Primer
10-mer
10-mer tail
AP1
AP2
AP3
5′-GCCATCGACC-3′
5′-GTCTACCAGGCATTCGCTTCATGCCATCGACC-3′
5′-GTCTACCAGGCATTCGCTTCATIIIIIGCCATCGACC-3′
5′-GTCTACCAGGCATTCGCTTCATIIIIIAGGAGATGCG-3′
5′-GTCTACCAGGCATTCGCTTCATIIIIICTCCGATGCC-3′
ACP, annealing control primer.
The polydeoxyinosine [poly(dI)] linkers are underlined. I represents deoxyinosine.
Vol. 36, No. 3 (2004) BioTechniques 3
which results in numerous nonspecific
products. Current differential display
methods have problems of the anchor/
anchor products or arbitrary/arbitrary
products. However, the ACP system
eliminated these problems because
PCR products were generated by only
the combination of oligo(dT)15 ACP
and arbitrary ACP, but not by either
oligo(dT)15 ACP or arbitrary ACP
alone (Figure 1, lanes 7–9).
Identification of Genes Expressed
Differentially During Mouse
Conceptus Development
GeneFishing technology was used
to detect genes that are expressed dif-
ferentially during mouse conceptus de-
velopment by using total RNAs isolat-
ed from 4.5 to 18.5 dpc [embryonic day
(E); E4.5–E18.5] mouse conceptus tis-
sues. From the differential expression
levels of mRNA fragments as observed
on agarose gels, 10 DEGs (DEG1–10)
were isolated from E4.5, E11.5, and
E18.5 tissues using 3 arbitrary ACPs
(Figure 2A). The expression patterns
of the DEGs were confirmed by North-
ern blot analysis (Figure 2B). Northern
blot analysis showed a different ex-
pression pattern for DEG10 from the
results seen on agarose gel because the
low resolution of the agarose gel did
not allow differentiation between two
different fragments, one of which was
DEG10, which were placed side by
side on the agarose gel.
The use of agarose simplifies the
process but generates low resolution.
Such a low resolution of agarose gel
does not allow the PCR products of
similar size to be distinguished. How-
ever, as shown in Table 2, our approach
generates PCR products with 9- to
10-mer base pair matches when the
3′-end core sequence (10-mer) of arbi-
trary ACP hybridizes to the first-strand
cDNA. In contrast, the current differ-
ential display generates PCR products
with 6- to 8-mer base pair matches (10).
Accordingly, our approach minimizes
the possibility of accidentally generat-
ing PCR products of similar size.
Sequence analysis showed that 5
of these 10 DEGs were known genes
(Table 2). The other 5 DEGs have not
been characterized although their full-
length cDNA or expressed sequence
tag (EST) sequences were deposited
in a National Center for Biotechnology
Information (NCBI) database (http://
www.ncbi.nlm.nih.gov/) (Table 2).
Northern blot and sequence analyses
suggest that DEG2 is an isoform of β-
tropomyosin 2. EST analysis of DEG5
and DEG7 showed homology to calde-
smon 1 and U6 small nuclear RNA
(SnRNA)-associated spliceosomal-like
protein LSM6, respectively. However,
because the expression patterns of these
DEGs during these embryo develop-
mental stages have not been character-
ized in detail, further study is needed.
NCBI database searches using the
full-length sequence of the predicted
proteins products of DEG6 and DEG10
revealed that DEG6 has significant ho-
mology with human decidual protein
induced by progesterone, but that the
predicted protein product of DEG10
is not homologous with any protein
Figure 1. Comparison of conventional primer and annealing control
primer (ACP) systems in differential display method. The first-strand
cDNAs are synthesized from the total RNAs of mouse conceptus tissues
at 18.5 [days postcoitus (dpc)] by using either (lanes 1–3) oligo(dT)15,
(lanes 4–6) oligo(dT)15 tail, or (lanes 7–9) oligo(dT)15 ACP. A two-
stage PCR amplification was conducted using a single primer or a pair
of primers. Lane 1, oligo(dT)15; lane 2, 10-mer; lane 3, oligo(dT)15 plus
10-mer; lane 4, oligo(dT)15 tail; lane 5, 10-mer tail; lane 6, oligo(dT)15
tail plus 10-mer tail; lane 7, oligo(dT)15 ACP; lane 8, 10-mer ACP; lane
9, oligo(dT)15 ACP plus 10-mer ACP. M represents a 100-bp size mark-
er (generated by Forever 100-bp Ladder Personalizer; Seegene).
Figure 2. Results of GeneFishing PCR for the identification of differentially ex-
pressed genes (DEGs) during mouse conceptus development. (A) RNA finger-
printing on agarose gel. The two-stage PCR amplification was performed using 1 of
the 3 arbitrary 10-mer ACPs in combination with oligo(dT)15 ACP as indicated. Ar-
rows indicate DEGs during mouse conceptus development. MW represents a 100-
bp size marker generated by Forever 100-bp Ladder Personalizer. (B) Northern blot
analysis of the 10 DEG clones shows their expression patterns during mouse con-
ceptus development. Arrows indicate DEGs during mouse conceptus development.
The loading controls (the lower part of each panel) show each gel before blotting,
stained with ethidium bromide and photographed in UV light, and demonstrate the
presence of similar levels of 18S and 28S rRNA. E, embryonic day.
4 BioTechniques Vol. 36, No. 3 (2004)
SHORT TECHNICAL REPORTS
sequences in any available sequence
database. Interestingly, the correspond-
ing genomic sequence analysis of
DEG10 also showed no homologous
structures in any available genomic
sequence database, which included rat
genomic sequence databases.
The ACP system detects fewer
bands per reaction compared to cur-
rent differential display methods
(which generate 50–100 bands), which
is a drawback that can be overcome by
using more arbitrary ACPs. However,
generating fewer bands and using a
high concentration of dNTP (200 µM)
allows researchers to use ethidium
bromide-stained agarose gel to detect
differentially expressed products. Cur-
rent differential display methods use
insufficient amounts of starting mate-
rial and an insufficient concentration
of dNTP (2–5 µM) to detect different
banding patterns; these factors are also
responsible for the low reproducibility
of differential display results (11,12).
In addition, because the cDNA frag-
ments obtained from differential dis-
play are short (typically 100–500
bp) and correspond to sequences at
the 3′ end of the gene that primarily
represent the 3′ untranslated region,
the fragments do not usually contain
a large portion of the coding region.
Therefore, labor-intensive screening
of full-length cDNA is required, unless
significant sequence homology (infor-
mation for gene classification and pre-
diction of function) is detected.
Differential display methods gener-
ally use denaturing polyacrylamide gels
and radioactive detection techniques,
which restricts the use of these meth-
ods to laboratories with the appropriate
equipment. Relatively long exposure
times and difficulty in isolating inter-
esting bands from the polyacrylamide
gels are additional drawbacks of dif-
ferential display techniques. Although
nonradioactive differential display
methods have recently been described,
including silver staining (13), fluores-
cence-labeled oligonucleotides (14),
and the use of biotinylated primers (15)
and ethidium bromide-stained agarose
gels (16–19), these methods have met
with only limited success. GeneFishing
technology reaction products can be
detected on ethidium bromide-stained
agarose gel, and the results are repro-
ducible and reliable, all of which great-
ly increases the speed of DEG analysis
while avoiding the use of radioactivity
and expensive methods of detection.
Table 2. Sequence Alignment of DEGs with Known Full-Length cDNAs or ESTs
GenBank®
Accession No. Alignment of Arbitrary 10-mer ACP and Oligo(dT)15 ACP
Sequencesa Identity cDNA
DEGs
M87635 145 1018
5′-GCCATCGACC-3′-------------------------------------- poly(A) tail
5′-GCCATCGACC-3′--------------------------------------oligo(dT)15 ACP
β-Tropomyosin 2 Full-length
DEG1
AK003186 183 1024
5′-GCCATCGACC-3′--------------------------------------poly(A) tail
5′-GCCATCGACC-3′--------------------------------------oligo(dT)15 ACP
A new isoform of
β-Tropomyosin 2 Full-length
DEG2
NM_011570 1471 1711 2675
5′-GCCATCGACC-3′----------poly(A) region---------poly(A) tail
5′-GCCATCGACC-3′----------oligo(dT)15 ACP
Testis-derived
transcript Full-length
DEG3
NM_011619 714 1112
5′-GCCATCGACC-3′--------------------------------------poly(A) tail
5′-GCCATCGACC-3′--------------------------------------oligo(dT)15 ACP
Troponin T2 Full-length
DEG4
BB609848 210 710
5′-AGGAGATGCG-3′---------poly(A) region-----------
5′-AGGAGATGCG-3′---------oligo(dT)15 ACP
Homology to
caldesmon 1 EST
DEG5
BC031533 1093 1279
5′-AGGAGATGCG-3′-------------------------------------poly(A) tail
5′-AGGAGATGCG-3′-------------------------------------oligo(dT)15 ACP
Uncharacterized Full-length
DEG6
CB589336 373 614
5′-cGGAGATGCG-3′--------------------------------------poly(A)
5′-aGGAGATGCG-3′--------------------------------------oligo(dT)15 ACP
Homology to
LSM6 EST
DEG7
BC043018 85 769
5′-AGGAGATGCG-3′-------------------------------------poly(A) tail
5′-AGGAGATGCG-3′-------------------------------------oligo(dT)15 ACP
T-complex testis
expressed 1 Full-length
DEG8
NM_153064 1029 1608
5′-AGGAGATGCG-3′-------------------------------------poly(A) tail
5′-AGGAGATGCG-3′-------------------------------------oligo(dT)15 ACP
NADH
dehydrogenase
Fe-S protein
Full-length
DEG9
XM_129567 1826 2436
5′-CTCCGATGCC-3′--------------------------------------poly(A) tail
5′-CTCCGATGCC-3′--------------------------------------oligo(dT)15 ACP
Uncharacterized Full-length
DEG10
DEG, differentially expressed gene; EST, expressed sequence tag; ACP, annealing control primer.
a
Numbers above the sequence indicate the position (bolded) of the nucleotide sequences of each cDNA. The 1-bp mismatch be-
tween the arbitrary 10-mer of DEG7 and the target sequence is indicated by lowercase letters.
Vol. 36, No. 3 (2004) BioTechniques 5
ACKNOWLEDGMENTS
Y.-J.K., C.-I.K., and Y.-Y.G. contrib-
uted equally to this work.
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Received 23 September 2003; accepted
7 January 2004.
Address correspondence to Jong-Yoon
Chun, Seegene Life Science Laboratory,
142-21 Samsung-dong, Kangnam-gu, Seoul
135-090, South Korea. e-mail: chun@see-
gene.com
