Volume 16 Number 10 1988
Nucleic Acids Research
The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium
salt solutions: application to identifying recombinant DNA clones
Kenneth A.Jacobs*, Richard Rudersdorf, Suzanne D.Neill, Joseph P.Dougherty, Eugene L.Brown and
Edward F.Fritsch
Genetics Institute Inc., 87 Cambridge Park Drive, Cambridge, MA 02140, USA
Received January 27, 1988; Revised and Accepted March 16, 1988
ABSTRACT
In solutions of tetraalkylammonium salts the melting
temperature of oligonucleotide duplexes is independent of
nucleotide sequence and thus GC content. Data quantitating the
destabilizing effects of various mismatches in these solvents are
also presented. The results are in accord with theories on DNA
melting and establish conditions under which oligonucleotides can
be used as hybridization probes with predictable and controllable
specificity.
INTRODUCTION
Synthetic oligodeoxyribonucleotides are used as probes for
the isolation of specific clones, as templates for introducing
site-specific changes in DNA sequences, and for the diagnosis of
human genetic diseases (see ref. 1 for a review). Each of these
applications requires hybridization of the oligonucleotide to its
complementary sequence.
The specificity of strand pairing is
reflected in whether the oligonucleotide hybridizes only to its
perfectly complementary sequence or also to sequences to which it
is not perfectly matched.
If the specificity is not optimal,
identifying particular clones and making correct diagnoses
becomes more difficult.
High molecular weight DNA molecules can be annealed in
sodium chloride-sodium citrate solutions (SSC) with predictable
and controllable specificity because the influence of ionic
strength (2), base composition (3), and mismatches (4,5) on
thermal stability is known. If these parameters were known for
oligonucleotides, they could be used with equivalent
predictability. Unfortunately, if the hybridization solvent is
SSC, the melting temperature of an oligonucleotide duplex is
i IRL Press Limited, Oxford, England.
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highly dependent on nucleotide sequence (base stacking)
(6,7,8,9), a determinant which is averaged out in high molecular
weight DNA. The melting temperature of an oligonucleotide duplex
can be measured (10), but to do this prior to every experiment
for every oligonucleotide is tedious. As a substitute, the
empirical rule proposed by Suggs et ai (11) has been used to
establish hybridization temperatures when oligonucleotides are
used as probes (12,13).
Another difficulty is experienced in screening recombinant
DNA libraries with a mixture or pool of oligonucleotides in SSC.
In this instance the length of the oligonucleotides is constant,
but the individual compounds have different sequences. Thus,
there is no unique melting temperature for the pool. Since it is
not known which member of the pool matches the target sequence,
conditions that allow the oligonucleotide with the lowest melting
temperature (usually the lowest GC content) to hybridize
efficiently must be used.
These conditions allow the
oligonucleotides of higher GC content to form stable hybrids with
sequences to which they are not perfectly complementary. As a
consequence, optimal specificity of the hybridization reaction is
not attained.
To address these problems, we have studied the thermal
stability of oligodeoxyribonucleotide duplexes in solutions of
2.4M t e t r a e t h y l a m m o n i u m c h l o r i d e (TEAC1) and 3M
tetramethylammonium chloride (TMAC1). In these salts the melting
temperature of long, native DNA is known to be independent of
base composition (14). If this were true of shorter duplexes,
the melting temperature of these duplexes could be reliably
predicted, and probes consisting of oligonucleotides of different
sequences could be used with better precision.
Our results extend those of Itakura and colleagues who
earlier characterized the effect of single-base pair mismatches
on the thermal stability of oligonucleotide duplexes (10) and
showed the utility of using pools of oligonucleotides as probes
to screen recombinant DNA libraries (15,16). Wood et al. (17)
have also suggested that TMAC1 is useful in a variety of
circumstances. Using TMAC1 in the hybridization solvent, we
isolated the gene for erythropoietin-the first instance in which
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a clone
was i s o l a t e d
from
a mammalian genomic library by
screening with highly degenerate pools of oligonucleotides (18).
EXPERIMENTAL PROCEDURES
Synthesis of Oligonucleotides
Oligonucleotides were prepared on an Applied Biosystems
Model 380A DNA synthesizer with reagents and synthesis programs
from the same supplier. The crude DNA obtained after thiophenol
and ammonium hydroxide treatments followed by ethyl acetate
extraction was purified on a 2 0% polyacrylamide/7M urea gel. The
sequences of the oligonucleotides (in the 5' to 3' direction) are
listed in Table I. They were chosen because of length, base
composition and because we had available cloned DNA complementary
to those sequences. Each oligonucleotide is designated by a
number referring to its length in nucleotides and a letter to
distinguish it from other oligonucleotides of the same length.
Some oligonucleotides are designated by an additional number (eg.
16C and 16C-1) to indicate they are identical to the parent
compound except for the indicated substitution. Compounds 14A,
14B, and 26A are contained within 32A, compound 14E within 19F.
The other compounds have no common sequences.
Measurement of Thermal Stability
Two methods were used to determine the thermal stability of
oligonucleotide duplexes. Melting temperatures (Tm) were
determined by annealing equal moles of two complementary
sequences in the appropriate solvent. Profiles of absorbance as a
function of temperature were measured continuously at 260nm on a
Gilford 2600 spectrophotometer equipped with a thermal
programmer. The heating rate was l°C/min. The data are plotted
as the percent change in hyperchromicity as a function of
temperature. The midpoint of the thermal transition is taken as
T m . The solvents were 0.4M Na + (0.07M sodium phosphate buffer,
pH6.8) O.lmM EDTA or 3M TMAC1-0.1M sodium phosphate buffer pH6.8.
TEAC1 and TMAC1 were purchased from Eastman Kodak Co. or Aldrich
Chemical Co. and prepared as described (19,20).
For t h e second m e t h o d , r a d i o a c t i v e l y labeled
oligonucleotides were prepared by using T4 polynucleotide kinase
(New England Biolabs) and 7-32P-ATP (New England Nuclear Corp).
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The radioactive probe was hybridized to cloned DNA which had
previously been immobilized onto nitrocellulose (Schleicher &
Schuell) or nylon filters (Pall Corp.) (21). Following an
overnight hybridization, the filters were washed to remove nonhybridized probe.
Melting temperatures were then established as described
(10) . Each filter was submerged in one milliliter of solvent
(see below) and heated at a specific temperature for five
minutes. Subsequently, the solvent was withdrawn, replaced by a
second aliquot, and the filter was incubated at a higher
temperature. After covering an appropriate temperature range,
the remaining probe (usually less than 10% of the amount
initially hybridized) was removed with 0.1N sodium hydroxide.
The Cerenkov radiation was counted to determine the amount of
radioactivity (oligonucleotide) released during each incubation.
The data were summed to obtain a cumulative profile of
radioactivity released during the successive incubations and
plotted as the cumulative percent of duplex dissociated at each
temperature. The temperature at which 50% of the hybridized
oligonucleotide was released is designated as the irreversible
melting temperature (T^) because it is a kinetic and not an
equilibrium determination (6) . Repeated measurements yielded
values that agreed within 1-2°C. The solvents used were 2XSSC0.1% SDS, 2.4M TEAC1-0.05M TrisHCl pH8, or 3M TMACl-0.2% SDS
buffered with either 0.05M TrisHCl pH8 or 0.02M sodium phosphate
buffer pH6.8, with no difference observed between these two
buffers.
RESULTS
Thermal Stability of Oliqonucleotide Duplexes in BBC. TERC1. and
TMAC1
To study the e f f e c t s of TEAC1 and TMAC1 on the thermal
s t a b i l i t y of s h o r t DNA d u p l e x e s , we s y n t h e s i z e d t h e
oligonucleotides l i s t e d in Table I and measured the irreversible
melting temperature of duplexes between these compounds and
filter-bound DNA in 2XSSC, 2.4M TEAC1, or 3M TMAC1. At these
concentrations of TEAC1 and TMAC1 the melting temperature of high
molecular weight DNA ( i . e . greater than several hundred base
pairs) i s independent of base composition (14).
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TABLE I
Sequences of oligonucleotides^
Oliqo
Sequence
10A
GGCAGTAATT
14A
14B
14C
14D
14E
TTTTTGCAAACATC
TTGCCTCAGCATAG
AGCACGACAGAGTA
GATGCACACAAGAG
TGCTGGCCCTCTGG
16A
16B
16C
16C-1
16C-2
16C-3
16C-4
16D
16E
16F
TTCCAGAAAATGACAT
CACGACACTATTTTAT
GGTGTAAAGCCAACAC
19A
19B
19C
19D
19E
19F
TATAATTCAGGTAAATTGG
TTCTTGCATATTCATACAA
AAGCGCAATATTCTGGCAG
ACGTTGTAAAACGACGGCC
AAGGGCTGCAGGCTGCCTG
GGCGCTGCTGGCCCTCTGG
26A
GCCTCAGCATAGTTTTTGCAAACATC
32A
32A-1
32A-2
32A-3
TCCTTTGCCTCAGCATAGTTTTTGCAAACATC
GATTCTGGGGTCCAAG
CCTGCCTGGCTGTGGC
CCCAGCCGTGGGAGCC
—T
T
Q
T
T—G
G
T
Q
G—
1
Oligonucleotides are designated by a number referring to length
in nucleotides and by a letter to distinguish compounds of the
same length.
Dashes indicate identity with the parent compound
except for the indicated substitution.
Examples of melting curves for oligonucleotides 19A (26%
GC) , 19C (47% GC) , and 19F (79% GC) hybridized to their
complementary sequences are shown in Figure 1. The point at
which 50% of the probe eluted is designated T^.
In SSC the
difference in T^ between 19A and 19F is 18°C, a consequence of
the 53% GC difference.
In contrast in TEAC1 and TMAC1 the
difference is much less. Thus, in both salts the contribution of
base composition to duplex stability is effectively negated.
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100
? /°
/ X
SSC /
1°
"O 80
<D
p
•o
\\
c
CO
-i= 60
CO
7
/
CD
cn
o I
/ /
.E 40
CO
7 / /
1 o'/
20
/ pj
yr*
30
50
70
0
30
70
Temperature (°C)
Figure 1. Thermal denaturation curves of oligonucleotides 19A
(D), 19C (o), and 19F (x) in 2XSSC (left), 2. 4M TEAC1 (right), or
3M TMAC1 (right) . Radioactively labeled oligonucleotides were
hybridized to DNA immobilized on filters, and subsequently eluted
by heating in the different solutions.
The amount of
radioactivity eluted at each temperature was measured and is
plotted as the cumulative percent of the total radioactivity
released. Only two lines could be drawn through the TEAC1 data.
A summary of our results for oligonucleotides ranging from
10-32 bases is shown in Table II. For oligonucleotides 16 bases
long, the difference in T^ between the least stable and most
stable duplexes is 17°C in 2X SSC but only 3°C in 3M TMAC1.
Similarly for 19mers, these values are 20°C in SSC, 5°C in TMAC1,
and 0°C in TEAC1. Thus in both 2.4M TEAC1 and 3M TMAC1, the
dTj/dX GC (the change in T^ per mole fraction GC) is much less
than in 2X SSC. Since dTm/dXGC
0 in 2. 4M TEACL and 3M TMAC1
for high molecular weight DNA, and these experiments show that
dT^/dX GC is small for DNA duplexes as short as 16 nucleotides,
the result is likely to hold true for all intermediate sizes of
DNA duplexes.
Theoretical arguments suggest that the melting temperature
of low molecular weight DNA is inversely proportional to the
length of the duplex (8). This has been demonstrated for sheared
bacteriophage DNA (of length greater than 384 base pairs) (8) and
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TABLE II
Thermal Stability of Oliaonucleotide Duplexes 1
Oliqo
%GC
Na+
TMA+
TEA +
32
15
10A
40
14A
14B
14C
14D
14E
28
50
50
50
71
38
44
47
46
46
46
47
49
51
44
16A
16B
16C
16D
16E
16F
31
31
50
56
75
81
44
46
52
52
60
61
52
53
55
52
54
54
19A
19B
19C
19D
19E
19F
26
26
47
53
68
79
50
48
60
62
67
68
61
59
64
62
62
61
38
2 6A
42
63
70
46
3 2A
41
70
75
49
31
34
38
38
1
T h e m e l t i n g t e m p e r a t u r e (°C) of p e r f e c t l y complementary
duplexes in solvents containing various cations as determined by
elution from filters
for mixtures of oligo d(CA) and oligo d(GT) (ranging in size from
8 to a b o u t 400 base pairs) in 0.02M and 0.07M N a + (22). To
extend these measurements to short, random DNA sequences would be
difficult.
The design of the experiment would have to account
not only for the differences in stability between AT and GC base
p a i r s , b u t also for the d i f f e r e n c e in stability d u e to base
stacking.
A l t e r n a t i v e l y , this theory suggests a test for a
s o l v e n t in w h i c h T m is to be independent of base composition.
N a m e l y , f o r D N A duplexes of d i f f e r e n t length (L) and b a s e
composition, a plot of T m vs 1/L will yield a straight line.
In Figure 2 the T^ data from Table II are plotted in this
way.
A single straight line cannot be drawn through the TMAC1
d a t a , b u t the melting temperatures of these short DNA duplexes
increase monotonically w i t h t h e length of the d u p l e x e s .
In
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80
SSC
-
t
6 60
t
-
0
:
40
A
•
0.02
0.04 0.06
0.08 0.10
0.02
[Duplex Length]
0.04
0.06
0.08 0.10
-i
Figure 2. Thermal stability of oligonucleotide duplexes in 2XSSC
or 3M TMAC1. The T^ data from Table II are plotted against the
inverse of the length (in nucleotides) of the duplex.
contrast, in SSC the overlaps in T^ among the duplexes of
different lengths are considerable, suggesting that parameters
other than duplex length contribute significantly to thermal
stability.
The data support the proposition that these ions interact
with DNA at the level of a single base pair (14). However, for
duplexes less than 16 nucleotides the relative contribution of
sequence independent aspects to stability increases. For
oligonucleotides 14 bases in length, the difference in T^ between
the least stable and most stable duplexes in SSC and TMAC1 are 9°
and 7° respectively, values which are not appreciably different
(Table II).
A least squares fit to the data in figure 2 for
oligonucleotdes from 16-32 bases yields the equation
Ti = -682 x (IT1) + 97
where L i s the number of bases in the oligonucleotide and T^ i s
measured in °C. This equation predicts that the T^ of long DNA
i s 97 °C.
Since t h i s approximates the Tm for long DNA in
solution (14) after correcting for the 7-10 °C difference between
Tj,, and T^ ( 6 ) , the equation should a l s o be r e l i a b l e for
oligonucleotides longer than 32 bases.
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100 •
Temperature (°C)
Figure 3. Thermal denaturation curves of duplexes between
oligonucleotides 16C, 16C-1, or 16C-4 and the complementary
sequence to 16C. Profiles of absorbance and temperature were
measured in 0.4M Na + in a spectrophotometer.
Data were
normalized to the total change in absorbance.
Effect of Base Pair Mismatches on the Thermal Stability of
Oliqonucleotide Duplexes
We next determined the effect of non Watson-Crick base pairs
on thermal stabililty. Data for destabilization by GT base pairs
(which form two hydrogen bonds (23)) on the T^ of 32-base
duplexes in SSC, TMAC1, and TEAC1 are presented in Table III.
The change in T^ as a function of the percent incorrect base
pairs (in parentheses) is approximately the same in all these
solutions. Estimates of the destabilization of high molecular
weight DNA by incorrect base pairs range from 1-1.5°C per percent
mismatch (4,5) .
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TABLE III
Effect of Base Pair Mismatches on Thermal Stability1
Oliqo
Mismatch
Na+
TMA +
TEA*
32A
32A-1
32A-2
32A-3
_
GTX2
GTx4
GTx6
70
59 (1.8)
52 (1.4)
46 (1.3)
76
67 (1.4)
55 (1.7)
50 (1.4)
50
42 (1.3)
32 (1.4)
16C
16C-1
16C-2
16C-3
16C-4
_
GT
AG
AC
CT
63
56
58
51
52
67
59
62
57
57
(1.1)
(0.8)
(1.9)
(1.8)
(1.3)
(0.8)
(1.6)
(1.6)
1
The m e l t i n g temperature (°C) of duplexes containing the
indicated mismatches in solvents containing various cations. The
data for the compounds of 32A were determined by elution from
f i l t e r s and for the c o m p o u n d s of 16C by heating in a
spectrophotometer.
Data in parentheses are the reduction in
thermal stability per percent mismatch relative to the perfectly
complementary duplex.
A different experiment showing the effects of a GT and a CT
mismatch on the thermal stability of 16-base long oligonucleotide
duplexes in 0.4M N a +
is shown in Figure 3. The DNA duplex, in
solution, was heated in a sealed cuvette in a spectrophotometer.
The data are plotted as the percent change in hyperchromicity
over temperature.
The T,,, is determined by the midpoint of the
helix-coil transition.
Data from similar experiments comparing
GT, AG, AC, and CT mismatches in 0.4M N a + and 3M TMAC1 are listed
in Table III. In both solutions the least destabilizing mismatch
is an AG base pair, followed by a GT, and then either an AC or CT
base pair, similar to the order found by others (24) . These non
Watson-Crick base pairs are equally destablizing in both salts.
(The thermal stability of the 16C duplex is higher in Table III
than in Table II because the former value was determined in
solution at higher oligonucleotide concentrations.
Under these
conditions, reannealing competes with denaturation.)
DISCUSSION
We have measured the thermal stability of oligonucleotide
duplexes which differ in length and/or GC content and have found
results in concordance with those obtained with high molecular
weight
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DNA
duplexes.
In solutions containing N a + , duplex
Nucleic Acids Research
stability varies with GC content. In solutions containing TMA+
or TEA+, stability is length dependent but sequence independent.
This has implications for screening recombinant DNA libraries
(see below) . These results are also in accord with the
conclusions of Wood et al. (17) who measured the stability of
duplexes (which contained mismatches) of different length but
similar GC content.
We have also determined the contribution of mismatched base
pairs to helix destabilization. The decrease in stability is
equivalent in Na + , TMA+, and TEA+ and is in close agreement with
the data obtained with high molecular weight DNA. Interestingly,
not all mismatched base pairs are equivalent in TMA+. Although
it is a better solvent for measuring homology between DNA
sequences than Na + (25), TMA+ is not neutral by this criterion.
The data in Tables II and III can be used to select a
hybridization temperature that will yield stable duplexes
between an oligonucleotide and target sequences which are
perfectly complementary. The value of this is most apparent when
s c r e e n i n g r e c o m b i n a n t l i b r a r i e s with m i x t u r e s of
oligonucleotides. In SSC the hybridization conditions must be
chosen to permit the oligo with the lowest GC content to anneal.
These conditions permit sequences of high GC content to form
stable duplexes with sequences to which they are not perfectly
complementary, with the following consequences. Statistically,
considering a genome to be an infinitely large collection of
random sequences, the probability P of finding a sequence of
length L with k mismatches in a genome which is 50% GC is given
by
p
L-k = (0.25)L~k(0/75)k L!/[k!(L-k)!].
Thus, if a single 16mer were being used to screen a library, the
ratio of hybridizing sequences with one mismatch to sequences
with no mismatches is calculated to be 48:1. If two mismatches
are allowed by the hybridization conditions, the ratio is 1080:1.
In SSC, this exponential increase in the number of
hybridizing sequences cannot be avoided. In contrast,even with a
mixture of oligos, hybridization conditions can be determined
solely on the basis of length in either TMAC1 or TEAC1. These
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conditions can be chosen to select against sequences with
multiple mismatches without selecting against a perfectly matched
target sequence. In these salts, all of the oligonucleotides in
a pool will hybridize with equal efficiency to perfectly
complementary sequences and with reduced efficiency to sequences
to which they are not perfectly complementary, thereby reducing
the number of false positives and facilitating isolation of the
clone of interest.
We routinely screen recombinant DNA libraries in TMAC1. A
summary of the experimental details for this
particular
application may be useful.
The hybridization solution is 3M
TMAC1, 0.05M NaPO4 pH6.8, lmM EDTA, 5X Denhardt solution, 0.6%
SDS,
and 100 i g/ml d e n a t u r e d salmon sperm DNA.
The
oligonucleotides are labeled by kinasing with c-32P-ATP and used
at 1-2 x 1 0 6 cpm per ml of hybridization solution.
The
hybridization temperature is 5-10 °C below the Ti, determined by
referring to Table II or by applying the equation given in the
text. Hybridization is for 2-3 days. The filters are washed in
3M TMAC1, 0.2% SDS, 50mM TrisCl pH8.0 several times at room
temperature and for one hour at a temperature 10-15°C below the
T^. The filters are then washed once in 2X SSC, 0.2% SDS at room
temperature and autoradiographed. The darkest signals correspond
to the clones which are most complementary to the probe. TMAC1
is preferred to TEAC1 because the higher temperatures reduce nonspecific background.
Hybridizing in TMAC1 gives better
specificity than hybridizing in SSC and then washing in TMAC1.
Either nitrocellulose or nylon filters can be used, but the
former become fragile in TMAC1 and must be handled carefully.
TMAC1 can be used in any situation involving DNA-DNA
hybridization.
Employing this technique, we isolated the gene
for erythrypoietin from a human genomic library using only pools
of oligonucleotides as probes (18). The considerations described
above for screening libraries provide a starting point for
determining the optimum conditions for other experimental
applications.
ACKNOWLEDGEMENTS
We wish to thank John E. Brown, Darlene A. Vanstone and
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David A. Palmer for assistance in oligonucleotide synthesis, Paul
Schendel and David Hill for helpful discussions and a critical
reading of the manuscript, and our colleagues at Genetics
I n s t i t u t e who made many useful suggestions in reducing these
observations to practice.
*To whom correspondence should be addressed
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