Polymer supported synthesis of aminooxyalkylated oligonucleotides, and some applications in the fabrication of microarrays

Article abstract of DOI:10.1016/j.bmc.2009.06.038

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synt

Full text of DOI:10.1016/j.bmc.2009.06.038

Bioorganic & Medicinal Chemistry 17 (2009) 5442–5450
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Polymer supported synthesis of aminooxyalkylated oligonucleotides,
and some applications in the fabrication of microarrays
a,
D. Sethi a, S. Patnaik b, A. Kumar a, R. P. Gandhi c, K. C. Gupta a,d, , P. Kumar
*
*
a Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi 110 007, India
b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Fogarty Hall, University of Rhode Island, Kingston, RI 02881, USA
c Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110 007, India
d Indian Institute of Toxicology Research, M.G. Marg, Lucknow 226 001, U.P., India
a r t i c l e i n f o
a b s t r a c t
A new protocol has been described for solid phase preparation of 30- and 50-aminooxylalkylated oligonu-
cleotides using commercially available reagents. This involves attachment of linker 4 either with an
LCAA-CPG support via succinoylation followed by synthesis (30-aminooxyalkylated oligomers) or forma-
tion of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 50-amin-
ooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high
yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer
and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were
used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated
by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of con-
ditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully
used for detection of nucleotide mismatches and bacterial typhoid.
Article history:
Received 23 April 2009
Revised 18 June 2009
Accepted 19 June 2009
Available online 23 June 2009
Keywords:
Aminooxyalkyl-oligonucleotides
Solid phase synthesis
Microarray
Hybridization
Base mismatch
Disease diagnosis
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
sequencing, mutation detection and medical research. The technol-
ogy allows highly parallel throughput processing and screening of
During the past decades, solid phase synthesis methodology has
remained a proficient and preferred tool for preparation of unmod-
ified oligonucleotides and their modified analogues.1–5 Synthesis of
oligonucleotides containing unnatural 30-terminal groups has be-
come increasingly important for various biomedical applications,
since modifications at the 50-terminal preclude 32P-labeling of oli-
gonucleotides through 50-phosphorylation. In addition, in order to
incorporate modifications at the 50-terminal via solid phase phos-
phoramidite chemistry, the preparation of linker phosphorami-
dites is required, which is rather tedious and necessitates strictly
anhydrous and inert conditions. Modifications at the 30-terminal
can be easily carried out by using engineered solid supports.5,6
Therefore, there is an increasing interest in synthesis of oligonucle-
otides with functionalities such as aminooxy, carboxyl, or phos-
phate groups attached at the 30-terminal.
different biological samples (e.g., mRNA, cDNA and proteins).7–15
DNA microarrays are mostly based on hybridization of targets with
double-stranded DNA or single-stranded oligonucleotides (probes)
immobilized on a polymer support (usually glass), followed by
detection by a scanning process. Of the two established methods,
viz., on-chip synthesis and deposition method, the latter is the pre-
ferred route for the preparation of oligonucleotide microarrays for
routine applications, as it offers flexibility in respect of different
chemistries for attachment of probes on a surface of choice. Differ-
ent strategies of microarray fabrication involve tethering of differ-
ent functional groups containing oligonucleotides and their
analogues directly through use of a linker to the support bound
functionalities.16–32 Aminoalkylated oligonucleotides are generally
used for post-synthetic attachment of oligonucleotides to the solid
substrate owing to high nucleophilicity of the primary amino
groups under basic conditions. However, the aminooxy group is a
better nucleophile than the primary amino group. The enhanced
One important application of end-modified oligonucleotides is
their use in the fabrication of oligonucleotide microarrays. Re-
cently, microarrays have widely been used as research tools for
various applications such as gene expression analysis, DNA
nucleophilicity of the aminooxy groups is perhaps due to
a-ef-
fect,33 which refers to the increased nucleophilicity of a functional
group due to the presence of an adjacent atom with a lone pair of
electrons. The presence of oxygen atom adjacent to amino group
enhances the nucleophilicity of the aminooxy group. Additionally,
pKa of aminooxy groups is in the range of 5–6, as compared to ami-
no groups (pKa ꢀ 9), which keeps aminooxy groups unprotonated
* Corresponding authors. Tel.: +91 11 27662491; fax: +91 11 27667471.
E-mail addresses: kcgupta@igib.res.in (K.C. Gupta), pkumar@igib.res.in (P.
Kumar).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.038

D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
5443
even at a low pH. This has already been reported that in acidic pH,
the aminooxy-modified oligomers react more rapidly than their
amino counterparts with electrophilic substrates.34,35 Salo et al.36
have reported a synthesis of aminooxyalkylated oligonucleotides.
However, the method is quite tedious and requires an additional
deprotection step, which may also result in the modification of
nucleobases.
tide microarrays for disease diagnosis, we kept the following
points in view, viz., (a) the synthetic strategy should be simple
and straight-forward, (b) it should involve commercially available
reagents and chemicals, (c) synthesis and deprotection steps
should not deviate from the standard procedures, (d) immobiliza-
tion of modified oligonucleotides should not require any condens-
ing/coupling reagent, (e) hybridization and immobilization
efficiencies should be high enough to enable visualization of fluo-
rescent spots at low concentration, and (f) the constructed micro-
arrays should be able to discern base mismatches. We have thus
employed the commercially available aminooxyacetic acid as a
source of aminooxyalkyl moiety. The aminooxyalkyl group was
protected by base-labile Fmoc protecting group to obtain N-(9-flu-
orenylmethyloxycarbonyl)-2-aminooxyacetic acid, 2, in ꢀ78%
yield. This was coupled with 3-aminopropan-1,2-diol in the pres-
ence of N,N-diisopropylcarbodiimide, to obtain N-(2,3-dihydroxy-
propyl)acetamide-2-N-(9-fluorenylmethyloxycarbonyl) oxyamine,
3, in ꢀ88% yield. Subsequent reaction with 4,40-dimethoxytrityl
chloride resulted in the formation of compound 4, which was suc-
cinoylated and coupled to long chain alkylamine-controlled pore
glass (LCAA-CPG) to obtain the engineered polymer support, 5,
Here, we report a simple method for the synthesis of 30- and
50-aminooxyalkylated oligonucleotides and a methodology for
the construction of biochips for detection of bacterial diseases.
The synthetic route involves the use of inexpensive, commer-
cially available chemicals and reagents for preparation of func-
tionalized polymer support, and the deprotection step is
carried out using aqueous 30% ammonia solution. The resulting
aminooxyalkylated oligonucleotides are then used for fabrication
of oligonucleotide microarrays on an epoxylated glass surface.
The modified oligonucleotides were immobilized at pH 6 and 9
with high immobilization efficiency (ꢀ18% at pH 6 and ꢀ22%
at pH 9) and signal-to-noise ratio (ꢀ97). The hybridization effi-
ciency was found to be ꢀ32% at pH 6. The constructed micro-
arrays were then used for discrimination of base mismatches.
On being subjected to different pH and thermal conditions, the
microarrays showed sufficient stability. Subsequently, oligonu-
cleotide probe-based microarrays were prepared following the
projected chemistry for detection of typhoid. The projected
methodology was compared with the existing method (immobi-
lization of aminoalkylated oligonucleotides on epoxylated glass
surface) with respect to immobilization efficiency of oligonucle-
otides at pH 6 and 9.33,34
with aminooxy group loading of ꢀ29
lmol/g of support (Scheme
1). All the intermediates were characterized by mass and 1H
NMR spectra.
In order to demonstrate the utility of the polymer support 5 in
the synthesis of modified oligonucleotides bearing aminooxy func-
tionalities at 30-end, oligonucleotide sequences (Table 1) were syn-
thesized at 0.2 lmol scale following standard protocol on a
Pharmacia Gene Assembler Plus. The oligomer, NH2OCH2CON-
HCH2CH(OH)CH2-OPO32ꢁ-d(TTC GTC ATC AAG TAG TTC CT) was
also assembled using the standard 50-DMTr thymidine support
with O-succinate linkage for comparison. The coupling efficiency
per cycle based upon the released 4,40-dimethoxytrityl cation ex-
ceeded 98.7% in both cases.
2. Results and discussion
In designing a simple route for synthesis of 30-aminooxyalkyl-
terminal oligonucleotides and in the construction of oligonucleo-
O
O
O
HO
N
O
a
b, c
O
H
H2N
OH
FmocHN
OH
O
OH
N
Fmoc
1
2
H
3
d
O
O
O
HO
O
N
e, f
N
H
N
H
H
O
Fmoc
O
O
O
Fmoc
ODMT
N
ODMT
N
H
H
5
4
Cl
P
g,i
NC
N
NH2
O
O
N
P
N
OR
h, i
O
H
NC
R = oligomer sequence
= Polymer support
OH
O
O
N
H
7
O
N
H
Fmoc
ODMT
3'- / 5'-aminooxyalkylated
oligonucleotides
6
Scheme 1. Reagents and conditions: (a) FmocCl (1 equiv), Na2CO3 (1.25 equiv), dioxane; (b) NHS (1.2 equiv), DIPCI (1.2 equiv), THF; (c) 3-amino-1,2-propane diol (1.2 equiv);
(d) DMTrCl (1.2 equiv), pyridine; (e) succinic anhydride (2 equiv), DMAP (0.5 equiv), TEA (2 equiv), EDC; (f) HBTU (1 equiv), LCAA-CPG (500); (g) oligo synthesis; (h) tetrazole,
; (i) deprotection, cleavage.
d(NNN)n

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D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
Table 1
Oligonucleotide sequences synthesized, their deprotection conditions and yields
No.
Oligonucleotiode sequence
Deprotection conditions
O.D. at A254 nm
1
2
3
4
5
6
7
8
9
TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
d(TTC GTC TTC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
d(TTC GTC TTC AAG TAG GTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
d(TTT TTT TTT TTTTTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
NH2OCH2CONHCH2CH(OH)CH2-OPO32ꢁ-d(TTC GTC ATC AAG TAG TTC CT)
TET-d(AGG AAC TAC TTG ATG ACG AA)
TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH2CH2CH2CH2CH2NH2
TET-d(ACC GGT GGA TGT GGC TTC CTT G) F. primer
TET-d(TGG TCT GCA GCA CCT TTT GAA C) R. primer
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
Aq NH4OH, 16 h, 60 °C
20.6
26.4
21.1
23.2
20.3
24.3
21.5
24.2
22.7
23.6
20.5
25.4
10
11
12
d(ACC GGT GGA TGT GGC TTC CTT GTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (probe sequence)
d(CGT CGA GTC GGG CAT CTT GGT ATT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (control)
The deblocking of oligomers (modified as well as unmodified)
cence intensity. As the pH increased beyond 9, the fluorescence
intensity decreased possibly because of partial hydrolysis of the
epoxy functions at higher pH, whereas the lower attachment den-
sity at pH below 6 could be explained in terms of lower reactivity
of the partially protonated aminooxyalkyl functions. Likewise,
inadequate protonation of epoxide ring at pH 7 and 8 resulted in
inefficient reaction of aminooxy groups with epoxylated glass sur-
face. Therefore, the attachment of aminooxyalkyl-modified oligo-
nucleotides was performed at pH values 6 and 9 in rest of the
experiments.
was carried out by treatment with aq ammonia (30%) at 60 °C for
16 h. The one step process allows removal of all the protecting
groups (b-cyanoethyl from phosphate and acyl from nucleic bases)
together with the elimination of the Fmoc from aminooxy function.
The ammoniacal solutions were concentrated and the oligomers
desalted on RP-silica gel column. The crude oligomers thus ob-
tained were analyzed on RP-HPLC. In one of the experiments, an
oligomer, d(TTT TTT TTT TTT TTT TTT TT), was assembled on the
standard d(T) support and modified polymer support 5. After
deprotection with aq ammonia and desalting, both the oligomers
were analyzed on HPLC. A TET-labeled T20 oligomer was also pre-
pared on the modified polymer support. Figure 1A and B shows
the HPLC profiles of crude oligomers, d(TTT TTT TTT TTT TTT TTT
TT) prepared on the standard and modified polymer support 5,
respectively and Figure 1C shows the elution profile of a co-injec-
tion of both the oligomers. RP-HPLC elution of TET-labeled d(T20) is
depicted in Figure 1D. Further, these oligomers were characterized
by mass spectrometry.
Further, to arrive at the optimal time and temperature required
for the immobilization reaction, an oligomer sequence, TET-d(TTC
GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2
(1 lM), dissolved in the reaction buffers of pH 6.0 and 9.0 sepa-
rately, was spotted onto epoxylated glass slides in duplicates and
kept at 35 °C. Microslides were withdrawn at different time inter-
vals (i.e., 60, 120, 180, 210, 240, 270, 300, 360 and 600 min) and
subjected to usual washings and drying. Subsequently, the slides
were scanned under a laser scanner and the spots were quantified.
Similar study was carried out at 45 °C and 55 °C and the spots were
quantified using QuantArray software. It was observed that, at pH
6.0, the reaction between 30-aminooxyalkylated oligonucleotide
and epoxy functions was completed in 270 min at 45 °C (having
highest fluorescence intensity), while, at pH 9.0, the reaction took
240 min for completion at 45 °C (Fig. 3B). Deviating from 45 °C, the
fluorescence intensity decreased at both the pH which might be
due to either incomplete reaction (at 35 °C, Fig. 3A) or partial deac-
tivation of the surface-bound epoxy functions (at 55 °C, Fig. 3C).
Therefore, the subsequent experiments involving immobilization
reaction were carried out at 45 °C.
In order to examine the efficacy of the projected strategy
involving immobilization of aminooxy-oligonucleotides on to
epoxylated glass surface to construct oligonucleotide microarrays
(biochips) useful for disease diagnosis, several parameters were
studied in detail. Epoxy functions on the glass surface were gener-
ated following the standard silanization procedure using 3-
glycidyloxypropyltrimethoxysilane.32
To assess the quality of the epoxylated glass surface, a labeled
oligomer, TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2, was spotted randomly all over the surface of
an epoxylated glass microslide. After usual incubation and wash-
ings, the slide was visualized under a laser scanner. The fluores-
cence intensity of individual spots on the slide was measured
using QuantArray software. The average intensity of the spots after
background subtraction was found to be in the range of ꢀ7400–
7700 A.U., indicating a uniform epoxylation of the glass surface.
In order to find out the optimal pH conditions required for effi-
cient immobilization of aminooxyalkyl-modified oligonucleotides
onto epoxylated glass microslides, the attachment of the labeled
probe, TET-d(TTC GTC ATC AAG TAG TTC CT-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2, was carried out at pH ranging from 4 to 11,
as described in Section 4. After washings and drying, the slide
was subjected to laser scanning. The results showed that the spots
corresponding to pH 6 and 9 exhibited the highest fluorescence
intensity of ꢀ7760 and ꢀ9450 A.U., respectively (Fig. 2). This is be-
cause the aminooxy groups remain almost unprotonated at pH 6
but the epoxy oxygen gets protonated, which triggers an acid cat-
alyzed opening of epoxide ring by the reaction with nucleophilic
aminooxy groups. The enhanced nucleophilicity of aminooxy
Having optimized pH, time and temperature required for immo-
bilization of aminooxyalkylated oligonucleotides onto epoxylated
glass surface, threshold concentration of the probe required for
visualization of spots under a laser scanner was determined by
spotting manually a serially diluted (40, 20, 10, 5, 2.5, 1.0 and
0.5 lM) labeled oligomer, TET-d(TTC GTC ATC AAG TAG TTC CT)-
OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (dissolved in the reaction
buffers of pH 6.0 and 9.0), for 270 and 240 min, respectively. After
usual processing, the slide was visualized under a laser scanner
(Fig. 4A and B represents the scanned images). It was inferred from
the figures that the fluorescence intensity of the spots increased
with increasing the concentration of the oligomer sequence, that
is, density of the immobilized oligomer increases with the concen-
tration reaching a plateau with oligomer concentration of 20
and higher.
lM
The thermal stability of the constructed microarray was exam-
ined by subjecting the spotted microslide to 0, 10 and 20 cycles of
PCR-like conditions. The results showed that oligonucleotides
immobilized following the projected strategy were sufficiently sta-
ble (ꢀ10% and ꢀ8.9% decrease in intensity after 20 cycles at pH 6
groups under basic conditions (pH 9) due to
a-effect results in
the maximum immobilization, i.e. spots with the highest fluores-

D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
5445
11.518
mA
11.02
mAU
1400
12.320
mA
A
B
C
1200
1000
1000
1200
1000
800
600
400
200
0
800
12.161
800
600
600
400
400
200
0
200
0
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
Time (min)
18.838
mA
500
D
400
300
200
100
0
0
5
10
15
20
Time (min)
Figure 1. HPLC elution profiles of (A) Crude d(TTT TTT TTT TTT TTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (m/z: 6246.8, M+); (B) crude d(TTT TTT TTT TTT TTT TTT TT)
(m/z: 6021.7, M+H+); (C) co-injection of d(TTT TTT TTT TTT TTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 and d(TTT TTT TTT TTT TTT TTT TT), and (D) TET-d(TTT TTT TTT
TTT TTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (m/z: 6927, M+Na+).
tent of accessibility of the surface-immobilized oligomer sequence
10000
for efficient hybridization, oligomer sequences, d(TTC GTC ATC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 and
7500
NH2OCH2CONHCH2CH(OH)CH2-OPO32ꢁ-d(TTC GTC ATC AAG TAG
TTC CT), were immobilized onto epoxyalkylated glass microslides
in duplicate at pH 6.0. After usual washings, the spots on the
microslides were exposed to complementary labeled oligonucleo-
tide sequence, TET-d(AGG AAC TAC TTG ATG ACG AA). The results
(Fig. 7) showed the hybridization efficiency to be almost the same
(ꢀ31.6%, 30-end immobilization vs ꢀ29.8%, 50-end immobilization)
irrespective of the nature of attachment of the probe sequences to
the glass surface. Therefore, 30-modified oligomers were employed
in the study as these can easily be assembled on the engineered
polymer support without affecting the standard synthesis and
deprotection conditions.
5000
2500
0
4
5
6
7
pH
8
9
10
Figure 2. pH study of immobilization of TET-d(TTC GTC ATC AAG TAG TTC CT)-
OPO3-CH2CH(OH)CH2NHCOCH2ONH2 onto epoxylated glass microslides.
The specificity and selectivity of the methodology was dem-
onstrated by immobilizing aminoalkyl- and aminooxyalkylated
oligonucleotides onto an epoxylated glass microslide at pH
6.0. The results of the experiment (Fig. 8) revealed that, under
acidic environment, the aminooxyalkylated oligomer reacts effi-
ciently with the epoxy function due to lower pKa of the amino-
oxy function and are immobilized with high fluorescence
intensity, whereas, the aminoalkyl function, having pKa ꢀ 8,
may get protonated and thus result in a diminished reaction
with surface-bound epoxy groups (which yield spots with lower
fluorescence intensity). Therefore, at pH 6, the aminooxyalkylat-
ed ligands could be immobilized selectively on the epoxylated
surface.
and 9, respectively) and thus can be used in biological research.
Figure 5 shows fluorescence intensity of untreated and heat-trea-
ted spots.
Likewise, stability to pH was evaluated by exposing the spotted
microarrays to buffers of different pH (7, 8 and 9) for 30 min each.
Figure 6 shows the results of this study, which indicated that fluo-
rescence intensity decreases by ꢀ3% at pH 8.0 whereas, at pH 9.0, it
decreases by ꢀ8%, as compared to fluorescence intensity observed
at pH 7.0.
The practical applicability of the above methodology was as-
sessed by hybridization experiments. In order to determine the ex-

5446
D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
10000
8000
6000
4000
2000
0
A
B
10000
8000
6000
4000
pH 6
pH 9
pH 6
pH 9
2000
0
0
200
400
600
0
200
400
600
Time (min)
Time (min)
10000
8000
6000
4000
2000
0
C
pH 6
pH 9
0
200
400
Time (min)
600
Figure 3. Graphs representing time kinetics to determine optimal time required for immobilization of aminooxyalkylated oligomers, in reaction buffers of pH 6 and 9, onto
epoxy-functionalized glass microslides at different temperatures (A) 35 °C, (B) 45 °C, and (C) 55 °C.
20000
16000
12000
8000
4000
0
Lane 2 17.6
10.7 7.4
6.6
5.4
3.2 2.5
pH6
pH 9
A
B
Lane 1 0.5
1
2.5
5
10
20
40
Lane 2 22.3 13.2 11.0
8.9
5.6 3.5 2.6
Lane 1 0.5
1
2.5
5
10 20
40
I
II
III
Figure 4. Threshold concentration of oligonucleotide sequence, TET-d(TTC GTC ATC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2, required for fluorescence
Figure 6. A graphical representation showing pH stability of the probes immobi-
lized onto epoxylated glass slides at pH 6 (orange) and 9 (blue). Subsequently, the
slides were subjected to washings with the washing buffer of pH 7, 8 and 9 (at
ambient temperature) and fluorescence intensity of the spots was determined. I, II
and III indicate stability at pH 7, 8 and 9, respectively.
visualization at (A) pH 6 and (B) pH 9. Lane 1, probe concentration (
immobilization efficiency (%).
lM); lane 2,
pH6
pH9
20000
16000
12000
8000
4000
0
Lane 1
Lane 2
0
8
16
24
32
Hybridization Efficiency (%)
0
10
20
Figure 7. Fluorescence map after performing hybridization assay with labeled
oligomer, TET-d(AGG AAC TAC TTG ATG ACG AA), of epoxylated glass slide spotted
with, d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (Lane
1) and NH2OCH2CONHCH2CH(OH)CH2-OPO32ꢁ-d(TTC GTC ATC AAG TAG TTC CT)
(Lane 2).
No. of thermal cycles
Figure 5. A graphical representation showing thermal stability of immobilized
probe after 0, 10 and 20 cycles of PCR-like conditions (n = 3).

D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
5447
Lane 1
Lane 2
Lane 1
Lane 2
0
4000
8000
Fl. Intensity (A.U.)
12000 16000 20000
Figure 10. Detection of bacterial typhoid. Lane 1: probe sequence. Lane 2: control
probe (non-complementary).
Figure 8. Fluorescence map of hybridization assay performed on epoxylated glass
slide spotted with TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2 (Lane 1) and TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-
CH2CH2CH2CH2CH2CH2NH2 (Lane 2) at 5 lM concentration in reaction buffer of pH
6, with quantitative histogram of fluorescence intensity.
aminooxyalkyl groups can be incorporated in oligonucleotides and
the aminooxyalkylated oligomers can be immobilized selectively
with high immobilization efficiency as compared to aminoalkylat-
ed oligomer at pH 6.
In order to demonstrate the capability of this methodology for
detecting base mismatches, four oligomers, viz., d(TTC GTC ATC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2, d(TTC
GTC TTC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2,
d(TTC GTC TTC AAG TAG GTC CT)-OPO3-CH2CH(OH)CH2NHC-
OCH2ONH2 and d(TTT TTT TTT TTT TTT TTT TT)-OPO3-
4. Experimental
4.1. General
Reagents and chemicals used in the present investigation were
purified prior to their use. The reactions requiring anhydrous con-
ditions were carried out under a blanket of dry Argon gas. 3-Glyc-
idyloxypropyltrimethoxysilane (GOPTS), N-methylimidazole (NMI)
and virgin glass microslides were procured from Sigma–Aldrich
Chemical Co., St. Louis, MO. Tetrachlorofluoresceinyl-phospho-
ramidite (TET-phosphoramidite) was purchased from Applied
Biosystems Inc., Foster City, CA. Other ancillary reagents and
chemicals were locally sourced. 1H NMR spectra were recorded
on a Bruker Avance 300 spectrometer operating at 300 MHz and
the chemical shifts (d) were reported in ppm relative to TMS. Mass
spectra of the organic compounds were recorded on Waters KC455
TOF-MS and oligonucleotides on a SELDI-TOF (Surface enhanced
laser desorption ionization-time of flight) PBS-II, Ciphergen Biosys-
tems, Freemont, CA using an 8-well gold plated chip. 3-Hydroxypi-
colinic acid mixed with picolinic acid and diammonium citrate was
used as matrix for oligomer analysis. Reversed-phase HPLC was
performed on an Agilent 1100 series system fitted with a variable
PDA detector set at (260 8) nm, using Hypersil Gold C-18 column
from Thermo Electron Inc., USA. Elutions were carried out with
increasing gradient of MeCN. Solvent A: 0.1 M ammonium acetate,
pH 7.0; solvent B: acetonitrile; gradient: 0–35% B in 25 min; flow
rate: 1 ml/min.
CH2CH(OH)CH2NHCOCH2ONH2 (2.5 lM) with zero, one, two
mismatches and non-complementary with respect to target
sequence, were immobilized. On hybridization, the perfect match
gave the highest fluorescence followed by one and two mis-
matches with diminished fluorescence, while non-complementary
sequences did not light up at all (Fig. 9). The signal-to-noise ratio
was ꢀ97 as calculated from the signals obtained from complemen-
tary hybridized spots and non-complementary spots. The quantita-
tive data of the scanned image is depicted in Figure 9.
In order to construct a biochip for detection of bacterial typhoid,
a microarray was constructed using a unique probe sequence of the
gene and a non-complementary probe sequence as a control. The
spots were subsequently hybridized with the labeled PCR ampli-
cons. After usual washings and drying, the spots were visualized
under a scanner. Figure 10 gives pictorial representation of the
scanned image. While the spots of the probe sequence specific to
tyv gene of bacteria (Salmonella typhi) showed fluorescence, the
spots specific to non-complementary probe did not fluoresce at
all, signifying the specificity of the system for preparing biochips
for detection of bacterial typhoid in humans.
3. Conclusions
A simple and rapid method for the preparation of 30- and 50-
aminooxyalkylated oligonucleotides has been developed. These
oligomers were then used to construct biochips useful for the
detection of nucleotide mismatches and bacterial typhoid. The
methodology offers an added advantage in as much as the multiple
4.2. Buffers used
(i) Reaction buffer: 0.1 M sodium acetate was prepared by dis-
solving 2.72 g in 200 ml of MilliQ water (containing 10% DMSO).
(ii) Capping buffer: 0.1 M Tris buffer containing 50 mM ethanol-
amine, pH 8.
(iii) Washing buffer (SSC buffer): 125 mM sodium citrate con-
taining 750 mM sodium chloride, pH 7.
(iv) Hybridization buffer: 125 mM sodium citrate containing
1 M sodium chloride, pH 7.
Lane 1
Lane 2
Lane 3
Lane 4
4.3. Preparation of polymer support 5 and phosphoramidite 6
4.3.1. Synthesis of N-(9-fluorenylmethyloxycarbonyl)-2-
aminooxyacetic acid, 2
0
1000
2000
3000
4000
Fl. Intensity (A.U.)
To a pre-cooled aqueous solution (20 ml) of 2-aminooxyacetic
acid hemihydrochloride 1 (10 mmol, 2.185 g) and sodium carbon-
ate (25 mmol, 2.65 g) was added dropwise a solution of 9-fluore-
nylmethyl chloroformate (20 mmol, 5.174 g) in dry dioxane
(20 ml) at 10 °C over a period of 15 min. After complete addition,
the mixture was allowed to stir for 4 h at 35 °C. The completion
of the reaction was monitored by TLC and after removal of the sol-
vent, the residue was taken up in ethyl acetate (100 ml). To this
was added 5% citric acid solution (50 ml). The organic layer was
Figure 9. Fluorescence maps of nucleotide mismatch detection with quantitative
analysis of fluorescence intensity. Four oligonucleotide sequences, d(TTC GTC ATC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (Lane 1), d(TTC GTC TTC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (Lane 2), d(TTC GTC TTC
AAG TAG GTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (Lane 3), and d(TTT TTT
TTT TTT TTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (Lane 4), were immobi-
lized on glass microslide. After usual washings, the spotted microslide was
hybridized with TET-d(AGG AAC TAC TTG ATG ACG AA) and subjected to a laser
scanner for visualization of fluorescent spots.

5448
D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
collected and subsequently washed with saturated solution of NaCl
(2 ꢂ 10 ml) followed by drying over anhydrous Na2SO4. After filtra-
tion, the solvent was removed on a rotavapor to give a white com-
zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (0.2 mmol). The reaction mixture was swirled for 30 s
and long chain alkylamine-CPG (500 mg) was added. The resulting
suspension was stirred for 30 min at ambient temperature and
then recovered on a sintered disc glass funnel and washed with
DMF (3 ꢂ 10 ml) and diethyl ether (2 ꢂ 10 ml), respectively. The
support was dried in a vacuum desiccator and subjected to capping
(residual amino groups) with a solution consisting of Ac2O:TEA:N-
methylimidazole:DCM (1:1:0.4:6, v/v/v/v) for 30 min at rt. After
washings with DCM (3 ꢂ 10 ml) containing triethylamine (1%,
v/v), the functionalized polymer support, 5, was dried in vacuo.
The loading on the support was calculated following the standard
protocol.
pound
2
(4.82 g, yield, ꢀ78%). Mass calculated: 312; mass
observed: 313.2 (M+H)+.
4.3.2. Synthesis of N-(2,3-dihydroxypropyl)acetamide-2-N-(9-
fluorenylmethyloxycarbonyl) oxyamine, 3
To the compound 2 (5 mmol, 1.56 g), dissolved in dry THF, were
added N-hydroxysuccinimide (6 mmol, 0.7 g), 4-dimethylamino-
pyridine (1 mmol, 122 mg) and N,N-diisopropylcarbodiimide
(6 mmol). After stirring the mixture for 4 h at room temperature,
3-amino-1,2-propanediol (6 mmol, 546 mg) was added and the
reaction was stirred further for 3 h at room temperature. The sol-
vent was evaporated, the residue taken up in ethyl acetate
(50 ml), washed with aqueous 10% citric acid solution (2 ꢂ 10 ml)
and dried over anhydrous Na2SO4 to obtain a crude product, which
was purified by column chromatography using 1,2-dichloroethane.
The desired compound, 3, was obtained in ꢀ88% yields (ꢀ1.7 g)
and characterized by its mass and 1H NMR. Mass calculated:
386; mass observed: 409.1 (M+Na+).
4.3.5. Preparation of phosphoramidite reagent of 4
N-{2-Hydroxy-3-(4,40-dimethoxytrityloxy)-propyl}acetamide-
2-N-(9-fluorenylmethyloxy-carbonyl)-oxyamine,
4
(790 mg,
1.0 mmol) was dissolved in anhydrous acetonitrile (10 ml) and to
this was added N,N,N0N0-tetraisopropyl-2-cyanoethylphosphorami-
dite (2 mmol). To this mixture, a solution of tetrazole (1 mmol,
70 mg dissolved in 2.5 ml of acetonitrile) was added dropwise at
room temperature and the reaction was allowed to stir for 2 h.
After completion of reaction (as monitored on TLC), methanol
(1 ml) was added to quench the reaction. The solvent was evapo-
rated on a rotary evaporator, the resulting syrupy residue taken
up in ethyl acetate (25 ml) and washed with saturated sodium
chloride solution (2 ꢂ 10 ml). The organic phase was collected,
dried over anhydrous Na2SO4, filtered, and the solvent was re-
moved. The material was purified over silica gel column and the
desired material was eluted with hexane/ethyl acetate/triethyl-
amine (5:2:1). The fractions containing compound 6 were pooled
and concentrated on a rotary evaporator (ꢀ82% yield).
1H NMR (CDCl3) d: 2.6 (t, 2H, –NCH2), 3.5–3.9 (m, 3H, –OCH–,
–OCH2–) 4.1–4.65 (m, 5H, –CH2OCO–, C9H, –OCH2CO–) and
7.2–8.1 (m, 8H, Ar–H).
4.3.3. Synthesis of N-{2-hydroxy-3-(4,40-dimethoxytrityloxy)-
propyl}acetamide-2-N-(9-fluorenylmethyloxycarbonyl)-
oxyamine, 4
Compound 3 (770 mg, 2.0 mmol) was dried by co-evaporation
with anhydrous pyridine (25 ml) and taken up in dry pyridine
(25 ml). To this, DMAP (0.2 mmol, 24.4 mg) and DMTrCl (711 mg,
2.4 mmol) were added and the mixture was stirred overnight at
room temperature. On completion of the reaction (as monitored
on TLC), excess DMTrCl was hydrolyzed by the addition of metha-
nol (0.5 ml). The reaction mixture was concentrated in vacuo and
traces of pyridine were removed by co-evaporations with toluene
(2 ꢂ 25 ml). The syrupy material, thus obtained, was dissolved in
ethyl acetate (75 ml) and washed with saturated aqueous solutions
of NaHCO3 (2 ꢂ 25 ml) and NaCl (1 ꢂ 25 ml), respectively. The or-
ganic phase was dried over Na2SO4 and concentrated in vacuo to
yield the crude product, which was purified by column chromatog-
raphy using 1,2-dichloroethane containing triethylamine (1%), as
an eluent. The desired compound, 4, was obtained in ꢀ85% yield
(1.168 g) and characterized by its mass and 1H NMR. Mass calcu-
lated: 687; mass observed: 709 (M+Na)+.
4.4. Synthesis and purification of modified oligonucleotides
A
number of oligonucleotide sequences (Table 1) were
assembled on the engineered polymer support 5 (for 30-terminal
modification). For 50-end modifications, oligonucleotides were syn-
thesized, and the last coupling was performed with the phospho-
ramidite reagent, 6, in
a manner analogous to the normal
nucleoside phosphoramidite couplings. For comparison purposes,
oligomers were also synthesized on the unmodified polymer
supports. Synthesis was carried out on Pharmacia Gene Assembler
Plus at 0.2 lmol scale following the standard phosphoramidite
chemistry. After synthesis, the support bound oligonucleotides
were subjected to 30% ammonia treatment for 16 h at 60 °C.
Subsequently, the ammonical solutions were concentrated in a
speed vac. Analysis and purification of oligonucleotides was carried
out on Agilent 1100 series system.
1H NMR (CDCl3) d: 2.7 (t, 2H, –NCH2), 3.5–3.85 (m, 9H, 2 ꢂ
–OCH3, –OCH–, –OCH2–) 4.2–4.6 (m, 5H, –CH2OCO–, C9H,
–OCH2CO–) and 7.15–8.2 (m, 21H, Ar–H).
4.3.4. Preparation of engineered polymer support
4.3.4.1. Succinoylation of 4.
1.0 mmol), succinic anhydride (200 mg, 2.0 mmol), dry TEA
(280 l, 2.0 mmol) and DMAP (61 mg, 0.5 mmol) was dissolved in
A
mixture of
4
(687 mg,
4.5. Immobilization studies
l
4.5.1. Glass silanization
dry ethylene dichloride (5 ml) and stirred for 30 min at rt. The
reaction was monitored on TLC and, after completion, it was
quenched by the addition of MeOH (0.5 ml). The reaction mixture
was further diluted with EDC (20 ml) and washed successively
with cold 5% aqueous citric acid (2 ꢂ 15 ml) and saturated solution
of NaCl (2 ꢂ 15 ml). The organic phase was collected, dried over
anhydrous Na2SO4 and concentrated to obtain the title compound
(745 mg; 95% yield), which was characterized by its mass. Mass
(m/z) calculated: 785; observed: 785 (M+).
The glass slides were treated with 1 M NaOH for 1 h followed by
washing with MilliQ water (3 ꢂ 150 ml). The slides were treated
with 1.5 M HCl for 1 h followed by washings with MilliQ water
(3 ꢂ 150 ml). The slides were then dipped in absolute ethanol
(2 ꢂ 15 min) and dried. The silanization was accomplished using
GOPTS (2%, v/v) in toluene for 4–5 h at 50 °C. After washing and
drying, the slides were stored under Ar atmosphere.
4.5.2. Evaluation of glass surface for immobilization
To evaluate the homogeneity of the epoxylated glass surface,
the following experiment was performed. An oligomer sequence,
TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHC-
4.3.4.2. Attachment of the above hemisuccinate to LCAA-
CPG. Succinoylated compound 4 (158 mg, 0.2 mmol) was dis-
solved in anhydrous DMF (2.0 ml). To this was added 2-(1H-ben-
OCH2ONH2 (1 lM), dissolved in reaction buffer, pH 6, was spotted

D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
5449
manually on an epoxylated glass microslide at different locations
all over the slide and kept overnight at 45 °C. After the reaction,
the microslide was treated with a capping buffer (0.1 M Tris con-
taining 50 mM ethanolamine, pH 8.0) for 15 min at 50 °C and then
washed with the washing buffer (2 ꢂ 50 ml) followed by MilliQ
water (2 ꢂ 50 ml), and dried under vacuum. Subsequently, the
spots on the microslide were visualized under a laser scanner
and quantified.
The stability of the constructed microarrays was also evaluated
by spotting the microslides in a similar way at pH 6 and 9, and then
subjecting them to washings with the washing buffer of different
pHs (7–9) at ambient temperature. The microslides were scanned,
after usual washings and drying, under a laser scanner.
4.6. Hybridization studies, specificity of chemistry and base
mismatch detection
4.5.3. Determination of optimal pH required for
immobilization
4.6.1. Comparison of hybridization of 30-and 50-aminooxy-
oligonucleotides
In order to study the effect of pH on immobilization of amin-
ooxyalkylated oligomers on epoxylated glass slides, the attach-
ment of probe was carried out at different pH (4–11). A labeled
oligomer, TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2, was dissolved in reaction buffers having
In order to study the effect of attachment of probe sequence on
the accessibility to target sequence, two oligomers, d(TTC GTC ATC
AAG TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 and
NH2OCH2CONHCH2CH(OH)CH2-OPO32ꢁ-d(TTC GTC ATC AAG TAG
TTC CT), dissolved in the reaction buffer of pH 6.0, were spotted
onto epoxy-glass microslide and kept at 45 °C for 240 min. The
residual epoxy functions were capped by treating the glass surface
with a capping buffer, 0.1 M Tris containing 50 mM ethanolamine,
pH 8, for 15 min at 50 °C. After usual washings with the washing
buffer, the spots were subjected to hybridization with the labeled
complementary target, TET-d(AGG AAC TAC TTG ATG ACG AA), as
described above. After washings with the hybridization buffer
(2 ꢂ 50 ml), the slide was dried and subjected to visualization of
spots under a laser scanner and quantification.
different pH and spotted (0.5
ll) onto epoxylated glass slides at a
concentration of 1 M using a pipettmen followed by overnight
l
incubation at 45 °C. The slide was processed and visualized, as de-
scribed above.
4.5.4. Immobilization kinetics
The results of the above experiment was used to optimize the
time and temperature required to fix the 30-aminoxy modified
oligonucleotides on the epoxylated glass surface, TET-labeled
oligomer, TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2 (1
l
M) was dissolved in the reaction buffer of
4.6.2. Specificity of chemistry
pH 6. Microarrays were prepared by spotting the above solutions
for different time intervals (60, 120, 180, 210, 240, 270, 300, 360,
and 600 min) and were kept at 35 °C. After the reaction, the slides
were washed with the washing buffer (2 ꢂ 50 ml), followed by Mil-
liQ water (2 ꢂ 50 ml), and dried under vacuum. The microslides
were subjected to scanning under laser. Similarly, the immobiliza-
tion reaction was allowed to proceed at temperatures of 45 °C and
55 °C.
Two oligonucleotides sequences, viz., TET-d(TTC GTC ATC AAG
TAG TTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (labeled amin-
ooxyalkylated) and TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-
CH2CH2CH2CH2CH2CH2NH2 (labeled aminoalkylated) (5 lM), were
dissolved in reaction buffer of pH 6, spotted onto an epoxylated
glass microslide in duplicates and kept overnight at 45 °C. After
the reaction, the microslide was washed with the washing buffer
(2 ꢂ 50 ml), followed by MilliQ water (2 ꢂ 50 ml), and dried under
vacuum. Subsequently, the slide was visualized under a laser
scanner.
Likewise, the experiment was repeated at pH 9. The extent of
reaction was evaluated by plotting fluorescence intensity (A.U.)
with respect to time (min).
4.6.3. Detection of base mismatches
4.5.5. Determination of threshold concentration of modified
oligonucleotides required for visualization of fluorescence
spots on glass microslides
In an attempt to arrive at the threshold concentration of amin-
ooxyalkylated oligonucleotides required for fluorescence detection
The specificity of the system was demonstrated by immobiliz-
ing modified oligomers on epoxylated glass microslides. Four
oligonucleotides, viz., d(TTC GTC ATC AAG TAG TTC CT)-OPO3-
CH2CH(OH)CH2NHCOCH2ONH2, d(TTC GTC TTC AAG TAG TTC CT)-
OPO3-CH2CH(OH)CH2NHCOCH2ONH2, d(TTC GTC TTC AAG TAG
GTC CT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 and d(TTT TTT TTT
at pH 6 and 9, the reaction was carried out by spotting (0.5
ll) a
serially diluted (40, 20, 10, 5, 2.5, 1.0 and 0.5 M) TET-labeled oli-
l
TTT TTT TTT TT)-OPO3-CH2CH(OH)CH2NHCOCH2ONH2 (2.5 lM),
gomer, TET-d(TTC GTC ATC AAG TAG TTC CT)-OPO3-CH2CH(OH)-
CH2NHCOCH2ONH2, dissolved in reaction buffers of pH 6 and 9,
onto epoxylated glass microslides and allowed to react at 45 °C
for 270 min and 240 min, respectively. After the reaction, the glass
slides were washed with the washing buffer and MilliQ water.
The slides were dried and visualized under a laser scanner. The
immobilization efficiencies were determined using the standard
curve.
having zero, one, and two mismatches and non-complementary
probe, were spotted on a glass microslide and processed as de-
scribed above. Subsequently, the spots on the microslide were
hybridized with a complementary labeled TET-d(AGG AAC TAC
TTG ATG ACG AA) and kept at 45 °C for 1 h and then at room tem-
perature for 12 h. After washings with the hybridization buffer
(3 ꢂ 50 ml), the microslide was subjected to laser scanning fol-
lowed by quantification.
4.5.6. Thermal and pH stability
4.7. Detection of bacterial typhoid
In order to evaluate the thermal stability of immobilization,
microarrays were constructed by spotting a TET-labeled oligo-
PCR-amplification: The PCR reaction was performed in 25 ll PCR
mer sequence (2.5
l
M), as mentioned above, in the reaction
Eppendorf containing 0.25 mM dNTPs (of each dATP, dCTP, dGTP,
and dTTP), forward primer TET-d(ACC GGT GGA TGT GGC TTC
CTT G), and reverse primer TET-d(TGG TCT GCA GCA CCT TTT
buffers of pH 6 and pH 9, respectively. After usual washings,
the constructed microarrays were subjected to PCR-like condi-
tions for 0, 10 and 20 cycles in SSC buffer (pH 7). PCR param-
eters were as follows; 94 °C for 30 s (denaturation step), 54 °C
for 30 s (annealing step), 72 °C for 30 s (extension step). After
thermocycling, the slides were dried and visualized under laser
scanner.
GAA C) (0.4 lM of each), genomic DNA (50 ng/reaction), Taq poly-
merase (0.75 units), and MilliQ water. All PCR reactions were per-
formed on an MJ Research thermo-cycler using the following
profile: (1) 5 min at 95 °C (initial denaturation step); (2) 35 cycles
of 1 min at 95 °C (denaturation step), 40 s at 61.6 °C (annealing

5450
D. Sethi et al. / Bioorg. Med. Chem. 17 (2009) 5442–5450
step), 2 min at 72 °C (extension step); (3) 7 min at 72 °C (final
extension step); hold temperature: 4 °C. The labeled PCR-ampli-
cons, 503 bp fragment of tyv gene (S. typhi), were analyzed on aga-
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Authors gratefully acknowledge the financial support from the
Department of Biotechnology, New Delhi, India. One of the authors,
DS, acknowledges the University Grants Commission (UGC), New
Delhi, India, for the award of Senior Research Fellowship to carry
out his work. Authors are also thankful to Drs. S. Gupta and S.
Khare, National Institute of Communicable Diseases (NICD), Delhi,
India, for providing typhoid sample and facilities.