标题：Tm值
计算方法
核酸Tm值（解链温度）计算 
评价标准是核酸所吸收的光线量达到其所增加吸收260nm光线的量的两倍达到的温度，
当核酸达到Tm值时，其260nm吸收量可增加百分之四十（紫外吸收量随解体程度而增加，直至完全解体。 
长度为25mer以下的引物，Tm计算公式为：Tm = 4℃(G + C)+ 2℃(A + T)
对于更长的寡聚核苷酸，Tm计算公式为：
Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) – 600/size         
公式中，Size = 引物长度。
影响因素
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 &micro;M 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.