Methods and systems for estimating the melting temperature (TM) for polynucleotide molecules
View Patent ↗The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, particularly duplexes of oligonucleotides which may be used, for example, as primers or probes in PCR and/or hybridization assays. The invention also relates to methods and systems for designing and selecting oligonucleotide probes and primers having a predicted melting temperature which is optimized for such assays. To this end, algorithms and methods are provided for predicting the melting temperature of a nucleic acid having a predetermined sequence. These methods and algorithms estimate the melting temperature of a nucleic acid duplex under particular salt conditions. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of particular salt conditions on the melting temperature. As such, the methods and systems of the invention provide superior result compared to existing methods, which do not consider sequence dependent effects of changing salt conditions.
1. A method for estimating a melting temperature (T m ) for a polynucleotide at a desired ion concentration [X + ], said polynucleotide having a known G-C content value, ƒ(G-C), comprising:
(a) obtaining a reference melting temperature (T m 0 ) for the polynucleotide, said reference melting temperature being a melting temperature obtained or provided for the polynucleotide at a reference ion concentration [X + ] 0 ; and
(b) modifying the reference melting temperature by a logarithm of the ratio of said desired ion concentration to said reference ion concentration, said logarithm being multiplied by a coefficient which is a function of the G-C content value,
wherein the estimated melting temperature is calculated using the reference melting temperature.
2. A method for estimating a melting temperature (T m ) for a polynucleotide at a desired ion concentration [X + ], said polynucleotide having a known G-C content value, ƒ(G-C), comprising:
(a) obtaining a reference melting temperature (T m 0 ) for the polynucleotide, said reference melting temperature being a melting temperature obtained or provided for the polynucleotide at a reference ion concentration [X + ] 0 ; and
(b) modifying the reference melting temperature by an amount,
k
(
f
(
G
-
C
)
)
×
ln
[
X
+
]
[
X
+
]
0
in which the coefficient k(ƒ(G-C)) is a function of the G-C content value ƒ(G-C), wherein the estimated melting temperature is obtained by using the reference melting temperature.
3. The method of claim 2 , wherein the coefficient k has a value determined by the relation
k (ƒ( G - C ))= m ·ƒ( G - C )+ k 0 ; and
wherein a first coefficient, m and a second coefficient, k 0 , are optimized for predicting polynucleotide melting temperatures T m 0 .
4. The method of claim 2 , wherein the reference melting temperature T m 0 is used to calculate T m according to the formula:
T
m
=
T
m
0
+
k
×
ln
[
X
+
]
[
X
+
]
0
.
5. The method of claim 4 , wherein the coefficient k
k (ƒ( G - C ))= m ·ƒ( G - C )+ k 0 ;
and wherein a first coefficient, m and a second coefficient, k 0 are optimized for predicting polynucleotide melting temperatures T m 0 .
6. The method of claim 2 , wherein the reference melting temperature T m 0 is used to calculate T m according to the formula:
T
m
=
T
m
0
+
k
(
f
(
G
-
C
)
)
×
ln
[
X
+
]
[
X
+
]
0
+
b
×
(
ln
2
[
X
+
]
-
ln
2
[
X
+
]
0
)
wherein a coefficient b is optimized for predicting polynucleotide melting temperatures.
7. The method of claim 6 , wherein k is m·ƒ(G-C)+k 0 ; and wherein a first coefficient, m, a second coefficient, k 0 and a third coefficient b are optimized for predicting polynucleotide melting temperatures T m 0 .
8. The method according to claim 5 , wherein m is −3.22, k 0 is 6.39.
9. The method according to claim 7 , wherein m is −4.62, k 0 is 4.52 and b=−0.985.
10. The method of claim 2 , wherein the reference melting temperature T m 0 is used to calculate T m according to the formula:
1
T
m
=
1
T
m
0
+
k
(
f
(
G
-
C
)
)
×
ln
[
X
+
]
[
X
+
]
0
.
11. The method of claim 10 , wherein the coefficient k has a determined value by the relation kƒ(G-C))=m·ƒ(G-C)+k 0 ; and wherein a first coefficient, m and a second coefficient, k 0 are optimized for predicting polynucleotide melting temperatures.
12. The method of claim 2 , wherein the melting temperature is obtained from the reference T m 0 by utilizing the formula:
1
T
m
=
1
T
m
0
+
k
(
f
(
G
-
C
)
)
×
ln
[
X
+
]
[
X
+
]
0
+
b
×
(
ln
2
[
X
+
]
-
ln
2
[
X
+
]
0
)
wherein a coefficient b is optimized for predicting polynucleotide melting temperatures.
13. The method of claim 10 , wherein k is m·ƒ(G-C)+k 0 ; and wherein a first coefficient, m and a second coefficient, k 0 , and a third coefficient b are optimized for predicting polynucleotide melting temperature.
14. The method of claim 11 , wherein k 0 is −6.18×10 −5 ; m is 3.85×10 −5 .
15. The method of claim 13 , wherein k 0 is −3.95×10 −5 ; m is 4.29×10 −1 ; and b is 9.40×10 −6 .
16. The method of claim 2 , wherein the G-C content value is the fraction of the polynucleotide's nucleotide bases that are either guanine or cytosine.
17. The method of claim 1 , wherein the polynucleotide is DNA.
18. The method of claim 1 , wherein the polynucleotide ranges in length from about 2 to about 500 basepairs.
19. The method of claim 1 , wherein the polynucleotide ranges in length from about 5 to about 200 base pairs.
20. The method of claim 1 , wherein the polynucleotide ranges from about 10 to about 30 basepairs in length.
21. The method of claim 1 , wherein the reference melting temperature is experimentally determined.
22. The method of claim 1 , wherein the reference melting temperature is calculated from a theoretical model.
23. The method of claim 1 , wherein the reference melting temperature is obtained by utilizing a nearest neighbor model.
24. The method of claim 1 , wherein the reference ion concentration is 1 M.
25. The method of claim 1 , wherein the ion is a monovalent ion.
26. The method of claim 1 , wherein the ion is selected from the group consisting of the cations of sodium, lithium, potassium, rubidium, cesium and francium.
27. The method of claim 1 , wherein the desired ion concentration ranges between about 1 mM and about 5M.
28. The method of claim 1 , wherein the desired ion concentration ranges between about 10 mM and about 2M.
29. The method of claim 1 , wherein the desired ion concentration ranges between about 70 mM and about 1021 mM.
30. 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 .
31. 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 .