US2012123751A1PendingUtilityA1
Method for Estimating a Melting Temperature of a Nucleic Acid in Buffers Containing Magnesium Ions
Est. expiryJan 5, 2027(~0.5 yrs left)· nominal 20-yr term from priority
G16B 20/20G16B 25/20G16B 25/00G16B 20/00
56
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, in the presence of divalent cations, particularly duplexes of oligonucleotides which may be used as, for example, but not limited to primers or probes in PCR and/or hybridization assays. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of divalent cation salt conditions on the melting temperature.
Claims
exact text as granted — not AI-modified1 . A method for estimating a melting temperature, T m (X 2+ ), for a polynucleotide at a desired divalent cation concentration, [X 2+ ], and an optionally present monovalent cation concentration [Mon + ], said polynucleotide having a known G-C content value, f GC , comprising:
(a) obtaining a reference melting temperature, T m °, for the polynucleotide, said reference melting temperature being a melting temperature obtained or provided for the polynucleotide at a reference monovalent ion concentration, [Mon +0 ]; and (b) modifying said reference melting temperature, or reciprocal of said melting temperature, by one or more terms which are a function of fGC to determine the melting temperature of the polynucleotide at the desired monovalent and divalent cation concentrations.
2 . The method of claim 1 , wherein the said reference melting temperature or the reciprocal of said reference melting temperature is modified by a term which is a function of the fGC multiplied by a term comprising a logarithm of the divalent cation concentration.
3 . The method of claim 2 , wherein the reciprocal of the reference melting temperature is modified by adding a term comprising a+b ln [X 2+ ]+f GC ·(c+d ln [X 2+ ]), wherein each of the coefficients a, b, c, and d is optimized for predicting polynucleotide melting temperatures.
4 . The method of claim 3 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
1
T
m
(
X
2
+
)
=
1
T
m
°
(
Mon
0
+
)
+
a
+
b
ln
[
X
2
+
]
+
f
GC
·
(
c
+
d
ln
[
X
2
+
]
)
wherein each of the coefficients a, b, c, and d is optimized for predicting polynucleotide melting temperatures.
5 . The method of claim 2 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
1
T
m
(
X
2
+
)
=
1
T
m
°
(
Mon
0
+
)
+
a
+
b
·
ln
[
X
2
+
]
+
f
GC
·
(
c
+
d
·
ln
[
X
2
+
]
)
+
(
e
+
f
·
ln
[
X
2
+
]
2
·
(
N
bp
-
1
)
)
wherein N bp , is the number of base pairs in the polynucleotide, and wherein each of the coefficients a, b, c, d, e, and f is optimized for predicting polynucleotide melting temperatures.
6 . The method of claim 2 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
1
T
m
(
X
2
+
)
=
1
T
m
°
(
Mon
0
+
)
+
a
+
b
·
ln
[
X
2
+
]
+
f
GC
·
(
c
+
d
·
ln
[
X
2
+
]
)
+
e
+
f
·
ln
[
X
2
+
]
+
g
·
(
ln
[
X
2
+
]
)
2
2
·
(
N
bp
-
1
)
wherein N bp , is the number of base pairs in the polynucleotide, and wherein each of the coefficients a, b, c, d, e, f and g is optimized for predicting polynucleotide melting temperatures.
7 . The method of claim 6 , wherein the reciprocal of the reference melting temperature is further modified by adding one or more additional terms
q
·
(
ln
[
X
2
+
]
)
p
2
·
(
N
bp
-
1
)
wherein N bp , is the number of base pairs in the polynucleotide, wherein p is an integer, and q is a coefficient which is optimized for predicting polynucleotide melting temperatures, and wherein one or more such terms may be added to the formula, and wherein p and q may be unique for each additional added term.
8 . The method of claim 2 , wherein the reference melting temperature is modified by adding a term comprising a′+b′·ln [X 2+ ]+f GC ·(c′+d′·ln [X 2+ ]), wherein each of the coefficients a′, b′, c′, and d′ is optimized for predicting polynucleotide melting temperatures.
9 . The method of claim 8 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
T m ( X 2+ )= T m °+a′+b ′·ln [ X 2+ ]+f GC ·( c′+d ′·ln [ X 2+ ])
wherein each of the coefficients a′, b′, c′, and d′ is optimized for predicting polynucleotide melting temperatures.
10 . The method of claim 2 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
T
m
(
X
2
+
)
=
T
m
°
+
a
′
+
b
′
·
ln
[
X
2
-
]
+
f
GC
·
(
c
′
+
d
′
·
ln
[
X
2
+
]
)
+
e
′
+
f
′
·
ln
[
X
2
+
]
2
·
(
N
bp
-
1
)
wherein, N bp is the number of base pairs in the polynucleotide, and wherein each of the coefficients a′, b′, c′, d′, e′, and f′ is optimized for predicting polynucleotide melting temperatures.
11 . The method of claim 2 , wherein the melting temperature, T m (X 2+ ), is estimated according to a formula comprising:
T
m
(
X
2
+
)
=
T
m
°
+
a
′
+
b
′
·
ln
[
X
2
+
]
+
f
GC
·
(
c
′
+
d
′
·
ln
[
X
2
+
]
)
+
e
′
+
f
′
·
ln
[
X
2
+
]
+
g
′
·
(
ln
[
X
2
+
]
)
2
2
·
(
N
bp
-
1
)
wherein N bp , is the number of base pairs in the polynucleotide, and wherein each of the coefficients a′, b′, c′, d′, e′, f′, and g′ is optimized for predicting polynucleotide melting temperatures.
12 . The method of claim 11 , wherein the reference melting temperature is further modified by adding one or more additional terms
q
·
(
ln
[
X
2
+
]
)
p
2
·
(
N
bp
-
1
)
wherein N bp , is the number of base pairs in the polynucleotide, wherein p is an integer, and q is a coefficient which is optimized for predicting polynucleotide melting temperatures, and wherein p and q may be unique for each additional added term.
13 . The method of claim 6 , wherein the coefficients a, b, c, d, e, f, and g are 3.92×10 −5 K −1 , −9.11×10 −6 K −1 , 6.26×10 −5 K −1 , 1.42×10 −5 K −1 , −4.82×10 −4 K −1 , 5.25×10 −4 K −1 , 8.31×10 −5 K −1 respectively and wherein the reference monovalent ion concentration, [Mon +0 ], is about 1 M.
14 . The method of claim 11 , wherein the coefficients a′, b′, c′, d′, e′, f′, and g′ are −4.59 K, 1.06 K, −7.26 K, −1.34 K, 63.3 K, −60.4 K, and −8.78 K respectively, and wherein the reference monovalent ion concentration, [Mon + 0 ], is about 1 M.
15 . The method of claim 2 , further comprising:
(a) calculating the ratio R of free divalent ion, [X 2+ ], and monovalent ion, [Mon + ], concentrations,
R
=
[
X
2
+
]
[
Mon
+
]
;
and
(b) comparing the ratio R to one or more limiting values.
16 . The method of claim 15 , wherein R is compared to the limiting values 0.22 M −1/2 and 6.0 M −1/2 .
17 . The methods of claims 6 , and 11 wherein the coefficients are allowed to vary with monovalent cation concentration, [Mon + ].
18 . The methods of claims 6 and 17 , wherein the coefficient a is calculated according to formula a=3.92×10 −5 (1−0.157−0.352√{square root over ([Mon + ])}·ln [Mon + ]), the coefficient d is calculated according to formula d=1.42×10 −5 (1+0.279−4.03×10 −3 ln [Mon + ]−8.03×10 −3 ln 2 [Mon + ]), the coefficient g is calculated according to formula g=8.31×10 −5 (1−0.514−0.258 ln [Mon + ]+5.25×10 −3 ln 3 [Mon + ]), and wherein the reference monovalent ion concentration, [Mon + 0 ], is about 1 M.
19 . The method of claim 1 , wherein the monovalent ion is selected from the group consisting of Tris ions, ammonium ions (NH 4 + ), lithium ions (Li + ), sodium ions (Na + ), potassium ions (K + ), rubidium ions (Rb + ), cesium ions (Cs + ), and francium ions (Fr + ).
20 . The method of claim 1 , wherein the divalent ion is selected from the group consisting of magnesium ions (Mg 2+ ), manganese ions (Mn 2+ ) and calcium ions (Ca 2+ ).
21 . The method of claim 1 , wherein the polynucleotide is DNA.
22 . The method of claim 1 , wherein the polynucleotide ranges in length from about 8 to about 500 base pairs.
23 . The method of claim 22 , wherein the polynucleotide ranges from about 10 to about 30 base pairs in length.
24 . The method of claim 1 , wherein the reference melting temperature is experimentally determined, calculated from a theoretical model, and/or calculated from a nearest neighbor model.
25 . The method of claim 1 , wherein the reference ion concentration is about 1 M.
26 . The method of claim 1 , wherein the divalent cation concentration ranges between about 0.1 mM and about 1 M.
27 . The method of claim 1 , wherein the presence of compounds that bind to the divalent cation is subtracted from the divalent cation concentration through the formula:
[ X 2+ ]=c ( X 2+ )− c (binding compound)×(no. of X 2+ ions bound per binding compound).
28 . A computer system for predicting a melting temperature, which computer system comprises:
(a) a memory; and (b) a processor interconnected with the memory and having one or more software components loaded therein, wherein the one or more software components cause the processor to execute steps of a method according to claim 1 .
29 . A computer program product comprising a computer readable medium having one or more software components encoded thereon in computer readable form, wherein the one or more software components may be loaded into a memory of a computer system and cause a processor interconnected with said memory to execute steps of a method according to claim 1 .Cited by (0)
No later patents cite this yet.
References (0)
No backward citations on record.