核酸Tm值（解链温度）计算
A variety of factors affect the efficiency of hybridization between two strands of DNA. These include the nature of the hybridizing molecules (DNA or RNA), their lengths, the hybridization environment (salt concentrations and denaturants), probe concentrations, and their sequences.
For membrane bound targets and moderately long DNA probes, Howley et al1 determined that the melting temperature (Tm) at which 50% of a probe is annealed to its complementary strand is defined by:
Tm = 81.5 + 16.6logM + 41(%G + %C) - 500/L - 0.62F
where
M = molar concentration of monovalent cations
%XG or C = the respective fraction of G and C nucleotides in the probe
L = length of the annealed product
F = molar concentration of formamide
For example, a short oligonucleotide probe with the sequence AGGTCATTG in a 75 mM solution without formamide has a predicted Tm = 81.5 + 16.6log(0.075) + 41(0.33+0.11) - 500/9 - 0.62(0)
= 24.1°C
This equation is inappropriate for probes less than about 50 nucleotides. Modifications of this equation include,
Tm of RNA = 79.8+18.5logM+58.4(%G+%C)+11.8(%G+%C)2-820/L-0.35F
Tm of an RNA-DNA hybrid = 79.8+18.5logM+58.4(%G+%C)+11.8(%G+%C)2-820/L-0.50F
The larger numbers reflect the increased stability of hybrids formed with RNA.
For oligonucleotides, Wallace, et al2 determined that
Td=2(A+T)+4(C+G), where Td = temperature (in °C) at which 50% of the oligonucleotides are annealed to their membrane-bound complementary sequences. The number of each particular nucleotide in the probe is inserted into the equation in place of the letters. The equation is useful for short (14-20 mers) in 0.9 M NaCl.
ex: for sequence AGGTCATTG, the Td = 2(2+3)+4(1+3) = 26°C
When the target and probe are free in solution, add 7-8°C to Td.
Melting temperature in solution is determined by plotting O.D versus temperature. The mid point on the S-shaped curve is the melting temperature.
Other estimates of melting points have been determined for DNA3 or RNA4 based on nearest neighbor analysis (reviewed by Genosys5). Breslauer, et al3 showed that melting behavior of a DNA duplex is predictable from its primary sequence.
Here,
Tm = 1000(DH)/[A+DS)+Rln(Ct/4)]-273.15+16.6log(Na+)].
where
DH = the sum of nearest neighbor enthalpy changes moving one base at a time through the sequence
A = correction for initiation of pairing (= -10.8)
DS = the sum of nearest neighbor entropy changes
R = 1.987 cal deg-1 mol-1)
Ct is the molar concentration of strands.
For self-complementary strands, the term "Ct/4" is replaced by Ct.
The values for DH and DS are shown in the table.
Nearest Neighbor DH DNA (kcal/mol) DH RNA (kcal/mol) DS DNA (cal/mol)DNA DS RNA (cal/mol)
AA or TT - 9.1 - 6.6 -24.0 -18.4
AT - 8.6 - 5.7 -23.9 -15.5
TA - 6.0 - 8.1 -16.9 -22.6
CA or TG - 5.8 -10.5 -12.9 -27.8
GT or AC - 6.5 -10.2 -17.3 -26.2
CT or AG - 7.8 - 7.6 -20.8 -19.2
GA or TC - 5.6 -13.3 -13.5 -35.5
CG -11.9 - 8.0 -27.8 -19.4
GC -11.1 -14.2 -26.7 -34.9
GG -11.0 -12.2 -26.6 -29.7
As an example, a 1 µM solution of the probe mentioned above (AGGTCATTG) in a 150 mM solution has a predicted Tm of:
Tm = 1000(-7.8-11.0-6.5-5.6-5.8-8.6-9.1-5.8) / [-10.8+(-20.8-26.6-17.3—13.5-12.9-23.9-24.0-12.9) + 1.987ln[(1E-06)/4]] - 273.15 + 16.6log(0.15)
= [-60900 / (-10.8-151.9-30.2)] - 273.15 - 13.7
= (-60900/-192.9) - 286.9
= 29°C
The applicability of these equations to laboratory situations varies as additional components in the hybridization environment are altered. It is best to consider these predictions guidelines, since they may vary for particular sequences from empirically derived determinations.
Background Problems
Moderate background on filter hybridizations is common. They often can be reduced by washing in up to 7% SDS.
Tg：玻璃化温度； Tc：结晶温度； Tm：熔点； Td：分解温度。
Tb是脆化温度，是玻璃态时能发生强du迫高弹形变的最低温度；Tm是结晶聚合物的熔点，即结晶聚合物熔融的温度；Tg是玻璃化温度，是玻璃态向高弹态开始转变的温度；Tf是粘流温度，是指非结晶聚合物从高弹态向粘流态转变的开始温度。
扩展资料：
熔点是固体将其物态由固态转变（熔化）为液态的温度，缩写为m.p.。而DNA分子的熔点一般可用Tm表示。进行相反动作（即由液态转为固态）的温度，称之为凝固点。与沸点不同的是，熔点受压力的影响很小。而大多数情况下一个物体的熔点就等于凝固点。
在有机化学领域中，对于纯粹的有机化合物，一般都有固定熔点。即在一定压力下，固-液两相之间的变化都是非常敏锐的，初熔至全熔的温度不超过0.5~1℃（熔点范围或称熔距、熔程）。
参考资料来源：百度百科-熔点
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