Engineering, 2013, 5, 440-445
http://dx.doi.org/10.4236/eng.2013.510B090 Published Online October 2013 (http://www.scirp.org/journal/eng)
Substitution of Deoxyinosine as Universal Base in
Oligonucleotides for DNA Ligati on
Zhiliang Yu
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China
Received 2013
ABSTRACT
Oligonucleotides libraries have been developed for various applications, but the library size of oligonucleotide increases
dramatically with the addition of oligonucleotide length. To assess the possibility of shortening library size by using
universal base, deoxyinosine (dITP), the effect of single/multiple dITP substituted in oligonucleotide on ligation was
investigated. It was found that different pairs with dITP had different lig ation patterns and pairs with dITP at different
locations also s howed different ligatio n patterns. With the departure of substitu tion position from ligati on site, the liga-
tion yield increased. Single dITP substitution at ligation site did extremely hurt the ligation efficiency, except for I:C
pair. On the other hand, single substitution at two bases or more apart from ligation site, there is no obvious effect on
ligation. Multiple dITP substitutions can more or less affect the ligation, besides I:C pair. This research demonstrated
that dITP can be applied to reduce oligonucleotide library size after substitution.
Keywords: Deoxyinosine; Substitution; Universal Base; Ligation
1. Introduction
Oligonucleotide libraries have been developed for a num-
ber of applications: sequencing by hybridization and li-
gation, mutant diagnostics, gene expression analysis, and
identification of microorg anisms. The size of a library of
oligonucleotide increases dramatically with increasing
length of oligonucleotide, e.g. a library of all 8mers would
require 65,536 oligonucleotides. People have attempted
to reduce library size by using shorter length [1]. How-
ever, it has a number of shortcomings, namely low sta-
bility of hybrids, difficulties in discriminating terminal
mismatches and a wide variation in the stabilities of AT-
and GC-rich duplexes. It was shown that the successful
way to reduce library size and also compensate for these
observed shortcomings is to add universal bases that can
pair with any of the four natural bases [2]. Therefore, a
universal base, which could substitute for any of the four
natural bases, would be of great utility for manipulating
DNA.
Inosine occurs naturally in the wobble position of the
anticodon of some tRNRs, where it appears to pair with
adenosine, cytidine and uridine of the codon of mRNAs
[3,4]. 2’-deoxyinosine (dITP) is widely used as an ambi-
guous nucleoside in oligonucleotide probes for hybridi-
zation [5,6], and primers for PCR [7-9] and sequencing
[10]. It was also reported that dITP can be incorporated
into PCR products when dITP is added to a mixture of
three dNTPs at normal concentrations and the fourth
dNTP at lower concentration [11-14]. However, to date,
very little work has been reported about the universal
base substitution in oligonucleotides for ligation. It has
been shown that neither 3-nitropyrro le nor 5-nitropyrrole
is recognized by T4 polynucleotide ligase when present
at either 5’- or 3’-end of an oligonucleotide [15]. It was
found that when 3-nitropyrrole is near to the 3’terminus,
i.e. still within active site of ligase, it can be recognized
by Tth DNA ligase and causes enhanced ligation fidelity.
If 3-nitropyrrole was substituted at the second site from
the nick no ligation observed [16]. Apart from this nitro-
pyrrole, one of universal bases, there is no report to sys-
tematically investigate the dITP substitution for DNA
ligation. Nowadays, dITP residues can be commercially
and economically substituted in oligonucleotides at ter-
minal and/or internal sites (https://www.idtdna.com/), al-
lowing dramatically reducing the oligonucleotide library
size. As nothing is known about the dITP substitution for
ligation, in this study, the effect of single- and multiple-
dITP substitution at different positions on ligation was
investigated.
2. Materials and Methods
2.1. Oligonucleotides
The oligonucleotides were designed using Primer Prem-
ier 5.0 software, and synthesized by Integrated DNA

Z. L. YU
441
Technologies (IDT, Coralville, IA, USA). The required
modification with phosphorylation and/or dITP substitu-
tion was also performed by IDT, and the oligonucleotides
were delivered after PAGE or HPLC purification.
2.2. Hybridization and Ligation
Typically, oligonucleotide probes were buffered in 10
mM Tri s-HCl, 1 mM EDTA, pH8.0 solution and the con-
centration was calculated after UV-Vis measurement us-
ing spectrophotometer (Nanodrop 1000, Wilmingto n, DE,
USA). Unless otherwise stated, Hybridization reaction
was performed in a 10 μl reaction mixture containing 1
μM oligonucleotide mixture after denature at 95˚C for 5
min followed by slowly cooling down to 10˚C for hybri-
dization. Then DNA oligonucleotide s were l iga t e d at room
temperature for 30 min after addition of ligation buffer
[66 mM Tris-HCl, pH7.6/10 mM MgCl2/1 mM dithioth-
reitol/7.5% PEG 6000/1 mM ATP] and Quick DNA li-
gase (New England Biolabs, MA, USA) in a final vo-
lume of 20 μl. Ligation reactions were terminated by
heating at 65˚C for 20 min.
2.3. Polyacrylamide Gel Electrophoresi s (PAGE)
After ligation, r eaction solution was mixed with 6x loa d-
ing dye and then electrophoresed on a 10% PAGE at 200
mV for 45 min using TBE buffer. After electrophoresis,
DNA was stained with SYBR green І (Invitrogen, Carlsbad,
USA) and visualized.
3. Results
3.1. Basic Description and P rocedure
As shown in Figure 1, 23 mer single stranded oligonuc-
leotide TP23F was designed with a 17 bases yellow re-
gion, which is complementary to 17 mer 5’-phosphory-
lated single stranded oligonucleotide BS17 (5’-Phos BS17),
and a 6 bases green region designated by 0, −1, −2, −3,
−4 and −5, respectively, from 5’ to 3’ according to the
distance from the directly ligated site. To investigate the
effect of dITP substitution on ligation, the natural bases
at this green region will be substituted by dITP accor-
dingly for ligation, unless otherwise stated. After simple
hybridization between 5’-Phos BS17 and TP23F, phos-
phorylated h ybrid with 17 bps duplex DNA flanked by a
6 bases single overhand (in green) at 5’ was formed. 30
mer single stranded DNA SST30F with a 24 mer red
region and also a 6mer green region, which is designed to
be complementary to the green region of TP23F. As re-
quired, the natural bases at this green region of SST30F
will also be modified through dITP substitution accord-
ingly, unless otherwise stated. After hybridization of
SST30F with the above duplex DNA, the longer DNA
product with a 20 bps ds-region and a 24 bases ss-region
was formed through ligase treatment.
3.2. Single dITP Substitution at Different
Positions for Different Natural Nucleotides
To investigate the effect of single dITP substitute on li-
gation, first, SST30F was such designed that its green
region is poly(dC)6, poly(dT)6, poly(dA)6 or poly(dG)6
to pair with p oly(dG)6, po ly(dA)6, poly(dT)6 o r poly(dC)
6, respectively, at the green region of TP23F. After addi-
tion of 5’-Phos BS17, three natural oligonucleotides
without sub stitution w ere expec tedly ligated by Q uick T4
ligase as shown in lane 2 of Figure 2. Then, the natural
bases at the green region of SST30F were changed indi-
vidually with single dITP substitution at different sites
from position “−5” to “0” for different natural bases (C,
T, A or G). Since the green region of TP23F was fixed
with natural bases, after hybridization, I:G ( Figure 2(A)),
I:A (Figure 2(B)), I:T (Figure 2(C)) or I :C (F igu re 2(D))
pairing accordingly formed at the green region where dI
came from SST30F and natural bases from TP23F. As
shown in Figure 2, single dITP can be used to substitute
all four natural bases for ligation at different positions
from “−5” to “0”. But ligation yields changed with the
change of the distance between substituted base and li-
gated base. And different pairs between dITP and natural
bases had different patterns of ligation yield. For I:G pair
in Figure 2(A), there was no obvious effect of single
dITP su bstitution at positions from “−5” to “−2” on liga-
tion yield and then very marginal effect after one base
closer to ligated nucleotide. Finally, the ligation yield of
I:G pair at position “0” dramatically decreased. Compa-
ratively, very slight effect of dITP substitution on liga-
tion yield occurred for I:A pair at position “−3” in Fig-
ure 2(B) and I:T pair at position “−2” in Figure 2(C),
respectively, instead of position “−1” for I:G pair. Then
the ligation yield significantly dropped if dITP substitu-
tion was put at position “−2” or “−1” for I:A pair and
position “−1” for I:T p air. Like for I:G pair at position “0”
in Figure 2(A), the ligation yields of dITP substitution at
position “0” for both I:A and I:T pairs were also very
weak, indicating almost zero ligation. In contrast, I:C
pair in Figure 2(D) showed very high ligation yield no
matter where the substitution is. Even substitution at po-
sition “0” had high ligation yield.
3.3. Multiple dITP Substitutions at “−3”, “−4” or
“−5” Position for Different Natural
Nucleotides
3.3.1. Tripl e Substitutions at “−3”, “−4” and “−5”
Positions
The results in Figure 2 indicate that for all four natural
nucleotides there is no apparent effect of single substitu-
tion from position “−5” to “−3” on ligation yield. There-

Z. L. YU
442
Figure 1. Schematic presentation of ligation with dITP substitution.

Z. L. YU
443
Figure 2. Ligation yields derived from I:G pair (A), I:A pair (B), I:T pair (C) and I:C pair (D), respectively, where dI is at the
green segment of SST30F and paired natural bases (dNTPs) are at the green region of TP23F, at different positions from “−5”
to “0” (from lane 3 to lane 8, respectively) according to the distance from the ligated base. As expected, positive control (lane
2) with all components has the desired ligated product based on the 10 bps DNA ladder (lane M), and negative control (lane 1)
without DNA ligase doesn’t have the ligated DNA.
fore, the question arises : is it possible to make triple dITP
substitutions at positions “−3”, “−4” and “−5” for all
natural bases? To address this question, TP23F was such
designed that its green region from position “−5” to “0”
is poly(dI)3(dC)3, poly(dI)3(dT)3, poly(dI)3(dA)3 or poly
(dI)3(dG)3 to pair with poly(dG)6, poly(dA)6, poly(dT)6 or
poly(dC)6, respectively, at the green region of SST30F.
After formation of hybrid between TP23F and SST30F, 3
G:Is, 3 A:Is, 3 T:Is, or 3 C:Is will accordingly be formed.
As shown in Figure 3, both triple A:I and T:I substitu-
tions gave similar results where positive controls without
substitutions (−) showed ligated products but substituted
reactions (+) didn’t have desired products. In contrast,
both positive control and substituted reaction for triple
C:I pairs had nice target products, indicating DNA mo-
lecules with triple dITP substitutions at positions “−3”,
“−4” and “−5” can be effectively ligated by Quick DNA
ligase. Interestedly, G:I pairs had similar results as C:I
pairs, except that the ligated product in substituted reac-
tion from G:I pairs migrated faster than the ligated prod-
ucts from C:I pairs. Actually, based on the DNA ladder
(lane M) and control experiment without SST30F (data
not shown), it was indicated that the ligated product in
substituted reaction for triple G:I pairs was the 40 bps
two-BS17-TP23F concatenated molecule with 3 C:I pairs
followed by 3 I:C pairs in the 6 bps green region instead
of the desired product, SST30F-BS17-TP23F, further
indicating that triple C:I or I:C pairs can be effectively
ligate d by Quick DN A l i gase.
Figure 3. Effect of triple dITP substitutions on ligation.
3.3.2. D ouble Subs ti tu ti ons at “−3”, “−4” or “−5”
Position
Since triple substitutions for A:I, T:I and G:I pairs cannot
work using Quick DNA ligase, except for C:I pairs, double
substitutions at position “−3”, “−4” or “−5” for A:I, T:I
and G:I pairs may be achievable. Therefore TP23F was
designed in the same way as in triple substitutions expe-
riments, except that only two dITPs were put at two out
of three positions from “−3” to “−5” instead of three
dITPs. The results in Figure 4 showed that the combina-
tion of double substitutions at position “ −4” and “−5” out
of three combinations for all three types of pairs had the
highest ligation yield, but compared to the ligation with-
out any substitution, the ligation yield of this combina-
tion at position “−4” and “−5” was still much lower. This
observation tells us two things. First, the distance of first
substituted nucleotide away from the ligated site is criti-
cal, which totally agrees with the findings of single sub s-
titution experiments; second, compared to single substi-
tution or triple substitutions, after one more or less subs-

Z. L. YU
444
Figure 4. Effect of double dITP substitutions on ligation.
titution, the yield drops or enhances obviously, respec-
tively, indicating that substitution number is also crucial
for ligation yield with substitution. Moreover, the over all
order of ligation yield with double substitutions is A:I >
T:I > G:I.
3.4. Recognition of dITP by Different Ligases
To investigate the feasibility o f ligation with substitution
at position “0” by different ligases, the widely used li-
gases including Quick T4 ligase, normal T4 DNA ligase,
E.coli DNA ligase and Taq DNA ligase were applied to
ligate I:A pair at position “0”. As shown in Figure 5,
different ligases had different ligation efficiencies for liga-
tion reactions without substitution; Quick T4 ligase showed
the highest yield fo llowed by normal T4 DNA ligase and
E.coli DNA ligase; Taq DNA ligas e almost gave no liga-
tion due to its short overhang. In contrast, all ligases test-
ed in this study gave similar results with very low liga-
tion yield after dITP substitution. Since there is no dif-
ference in ligation yield with substitution among 4 ligas-
es, and Quick T4 ligase gave the highest ligation yield
without substitution, Quick T4 ligase was chosen for
subsequent further tests.
3.5. Increasing of Ligation Yield by Adding dITP
To investigate the feasibility of increasing the ligation
yield by adding dITP, TP23F was such designed that its
green region is poly(dT)6 to be complementary to poly
(dA)6 at the green region of SST30F. Since there is no
substitution, after ligation, it expectedly gave very nice
ligated band as shown in lane 2 of Figure 6. If poly(dT)6
of TP23F at the green region was shortened to poly(dT)4
with two bases reduction, accordingly the ligation yield
dropped remarkably as shown in lane 3 of Figure 6,
which is presumably due to the decrease of thermal sta-
bility. Then, if two dI TPs were add ed at 3’-end of TP23F
to enlarge its green region to be 6 bases again with a se-
quence of poly(dT)4(dI)2, the ligation yield was dramat-
ically recovered as shown in lane 4 of Figure 6. Howev-
er, if two dCs instead of two dITPs were used, the liga-
tion yield further decreased as shown in lane 5 of Figure
6, indicating that mismatch introduction can seriously
hurt the ligation. All these findings indicate two things: 1)
dITP can be used to increase the effective oligonucleo-
Figure 5. Recognition of dITP substituted in ol igonuc leotide
by ligase.
Figure 6. Increase of ligation yield after dITP substitution.
tide size for ligation; 2) multiple dITPs can be substituted
within the certain bases from the ligated site as found
above.
4. Conclusions
4.1. For Single Substitution
1) Different pairs w ith dITP had different ligation pa t-
terns: I:C pair had the highest ligation efficiency, fol-
lowed by I:G p a i r, I:T pair and I: A pa i r.
2) Substitution at different locations have different li-
gation patterns: dITP substitution directly at ligation site
did extremely hurt ligation yields for I:G pair, I:T pair
and I:A pair, except for I:C pair. With the departure of
substitution position from ligation site, the ligation in-
creased. dITP substitution at two bases or more apart from
ligation site had no effect on ligation.
4.2. For Multiple Substitutions
1) Triple substitutions: There was no ligation yield
with triple substitution s at positions “−3”, “−4” and “−5”
for I:G pair, I:T pair and I:A pair. However, ligation with
triple substitutions for I:C pair still work ed well.
2) Double substitutions: The combination of double
substitutions at position “−4” and “−5” out of three com-
binations for all three types of p airs had the highest lig a-
tion yield, but compared to the ligation without any subs-
titution, the ligation yield of this combination was still
much lower. Moreover, the overall order of ligation yield
with double substitutions is A:I > T:I > G:I.

Z. L. YU
445
4.3. All These Findings Indicate
1) dITP can be used to increase the effective oligonu-
cleotide size for ligation;
2) Multiple dITPs can substitute the certain bases from
the ligated site as found above.
5. Acknowledgements
This work was sup ported by Natural Science F oundation
of Zhejiang Province, China (Y5100153), Welfare Tech-
nology Applied Research Project of Zhejiang Province,
China (2011C23007), and Natural Science Foundation of
ZJUT (2010021 3) to Z.L. Yu.
REFERENCES
[1] A. V. Fotin, A. L. Drobyshev, A. N. Perov and A. D. Mir-
zabekov, “Parallel Thermodynamic Analysis of Duplexes
on Oligodeoxyribonucleotide Microchips,” Nucleic Acids
Research, Vol. 26, No. 6, 1998, pp. 1515-1521.
http://dx.doi.org/10.1093/nar/26.6.1515
[2] S. Parinov, V. Barsky, G. Yershov, E. Kirillov, E. Timo-
feev, A. Belgovskiy and A. Mirzabekov, “DNA Sequenc-
ing by Hybridization to Microchip Octa- and Decanucleo-
tides Extended by Stacked Pentanucleotides,” Nucleic
Acids Research, Vol. 24, No. 15, 1996, pp. 2998-3004.
http://dx.doi.org/10.1093/nar/24.15.2998
[3] F. H. Crick, “Codon-Anticodon Pairing: the Wobble Hy-
pothesis,” J ournal of Molecular Biology, Vol. 19, No. 2,
1966, pp. 548-555.
http://dx.doi.org/10.1016/S0022-2836(66)80022-0
[4] M. D. Topal and J. R. Fresco, “Base Pairing and Fidelity
in Codon-anticodon Interaction,” Nature, Vol. 263, No.
5575, 1976, pp. 289-293.
http://dx.doi.org/10.1038/263289a0
[5] E. Ohtsuka, S. Matsuki, M. Ikehara, Y. Takahashi and K.
Matsubara, “An Alternative Approach to Deoxyoligonu-
cleotides as Hybridization Probes by Insertion of Deox-
yinosine at Ambiguous Codon Positions,” Journal of Bi-
ological Chemistry, Vol. 260, No. 5, 1985, pp. 2605-
2608.
[6] Y. Kawase, S. Iwai, H. Inoue, K. Miura and E. Ohtsuka,
“Studies on Nucleic Acid Interactions. I. Stabilities of
Mini-Duplexes (dG2A4XA4G2-dC2T4YT4C2) and Self-
Complementary d(GGGAAXYTTCCC) Containing Deo-
xyinosine and Other Mismatched Bases,” Nucleic Acids
Research, Vol. 14, No. 19, 1986, pp. 7727-7736.
http://dx.doi.org/10.1093/nar/14.19.7727
[7] R. V. Patil and E. E. Dekker, “PCR Amplification of an
Escherichia coli Gene Using Mixed Primers Containing
Deoxyinosine at Ambiguous Positions in Degene rate Ami-
no Acid Codons,” Nucleic Acids Research, Vol. 18, No.
10, 1990, p. 3080.
http://dx.doi.org/10.1093/nar/18.10.3080
[8] H. Liu and R. Nichols, “PCR Amplification Using Deox-
yinosine to Replace An Entire Codon and at Ambiguous
Positions,” Biotechniques, Vol. 16, No. 1, 1994, pp. 24-
26.
[9] D. R. Kil patri ck, B. Nottay , C. F. Yang, S. J. Ya ng, M. N.
Mulders, B. P. Holloway, et al., “Group-Specific Identifi-
cation of Polioviruses by PCR Using Primers Containing
Mixed-Base or Deoxyinosine Residue at Positions of Co-
don Degeneracy,” Journal of Clinical Microbiology, Vol.
34, No. 12, 1996, pp. 2990-2996.
[10] D. Loakes, D. M. Brown, S. Linde and F. Hill, “3-Nitro-
pyrrole and 5-Nitroindole as Universal Bases in Primers
for DNA Sequencing and PCR,” Nucleic Acids Research,
Vol. 23, No. 13, 1995, pp. 2361-2366.
http://dx.doi.org/10.1093/nar/23.13.2361
[11] H. Dierick, M. Stul, W. De Kelver, P. Marynen and J. J.
Cassiman, “Incorporation of dITP or 7-Deaza dGTP dur-
ing PCR Improves Sequencing of the Product,” Nucleic
Acids Research, Vol. 21, No. 18, 1993, pp. 4427-4428.
http://dx.doi.org/10.1093/nar/21.18.4427
[12] S. L. Turner and F. J. Jenkins, “Use of Deoxyinosine in
PCR to Improve Amplification of GC-rich DNA,” Biote-
chniques, Vol. 19, No. 1, 1995, pp. 48-52.
[13] O. P. Kuipers, “Random Mutagenesis by Using Mixtures
of dNTP and dITP in PCR,” Methods in Molecular Biol-
ogy, Vol. 57, 1996, pp. 351-356.
[14] A. Kobayashi, M. Kitaoka and K. Hayashi, “Analyses of
PCR Products Using DNA Templates Containing A Con-
secutive Ceoxyinosine Sequence,” Nucleic Acids Sympo-
sium Series (Oxford), Vol. 48, 2004, pp. 225-226.
http://dx.doi.org/10.1093/nass/48.1.225
[15] D. Loakes, A. Van Aerschot, D. M. Brown and F. Hill,
“Enzymatic Recognition of Acyclic Universal Base Ana-
logues in Oligonucleotides,” Nucleosides & Nucleotides,
Vol. 15, No. 11-12, 1996, pp. 1891-1904.
http://dx.doi.org/10.1080/07328319608002740
[16] J. Luo, D. E. Bergstrom and F. Barany, “Improving the
Fidelity of Thermus thermophilus DNA Ligase,” Nucleic
Acids Research, Vol. 24, No. 15, 1996, pp. 3071-3078.
http://dx.doi.org/10.1093/nar/24.15.3071