核酸中影响Tm值的因素有哪些？ 影响Tm的变性剂，是使核酸变性的原因，还是使Tm改变的原因呢
\u4ec0\u4e48\u662f\u89e3\u94fe\u6e29\u5ea6\uff1f\u5f71\u54cd\u7279\u5b9a\u6838\u9178\u5206\u5b50tm\u503c\u5927\u5c0f\u7684\u56e0\u7d20\u662f\u4ec0\u4e48\u89e3\u94fe\u6e29\u5ea6\uff08Tm\uff09\u662f\u5f15\u7269\u7684\u4e00\u4e2a\u91cd\u8981\u53c2\u6570\uff0c\u5b83\u662f\u5f5350%\u7684\u5f15\u7269\u548c\u4e92\u8865\u5e8f\u5217\u8868\u73b0\u4e3a\u53cc\u94feDNA\u5206\u5b50\u65f6\u7684\u6e29\u5ea6\uff0c\u4e00\u79cdDNA\u5206\u5b50\u7684Tm\u503c\u5927\u5c0f\u4e0e\u5176\u6240\u542b\u78b1\u57fa\u4e2d\u7684G+C\u6bd4\u4f8b\u76f8\u5173\u3002
\u5f71\u54cd\u56e0\u7d20\uff1a
G+C\u6bd4\u4f8b\u8d8a\u9ad8\uff0cTm\u503c\u8d8a\u9ad8\u3002
Tm=4\uff08G+C\uff09+2\uff08A+T\uff09
Tm\u503c\u4e3aPCR\u53cd\u5e94\u9000\u706b\u6e29\u5ea6\u7684\u91cd\u8981\u53c2\u8003\u4f9d\u636e\u3002
\u901a\u5e38\u5c06\u52a0\u70ed\u53d8\u6027\u4f7fDNA\u7684\u53cc\u87ba\u65cb\u7ed3\u6784\u5931\u53bb\u4e00\u534a\u65f6\u7684\u6e29\u5ea6\u79f0\u4e3a\u8be5DNA\u7684\u7194\u70b9\u6216\u6eb6\u89e3\u6e29\u5ea6\uff08melting temperature\uff09\uff0c\u7528Tm\u8868\u793a\u3002\u5f53\u8fbe\u5230Tm\u65f6\uff0cDNA\u5206\u5b50\u518550%\u7684\u53cc\u94fe\u7ed3\u6784\u88ab\u6253\u5f00\uff0c\u5373\u589e\u8272\u6548\u5e94\u8fbe\u5230\u4e00\u534a\u65f6\u7684\u6e29\u5ea6\uff0c\u5b83\u5728S\u578b\u66f2\u7ebf\u4e0a\u76f8\u5f53\u4e8e\u5438\u5149\u7387\u589e\u52a0\u7684\u4e2d\u70b9\u5904\u6240\u5bf9\u5e94\u7684\u6a2a\u5750\u6807\u3002
\u5f71\u54cdTm\u7684\u53d8\u6027\u5242\uff0c\u6ca1\u6709\u542c\u8bf4\u8fc7\u3002
Tm\u662f\u7531DNA\u5e8f\u5217\u672c\u8eab\u51b3\u5b9a\u7684\u5427
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.
热变性一半时的温度称为熔点或变性温度，以Tm来表示。DNA的G+C含量影响Tm值。由于G≡C比A＝T碱基对更稳定，因此富含G≡C的DNA比富含A＝T的DNA具有更高的熔解温度。根据经验公式xG+C =（Tm - 69.3）× 2.44可以由DNA的Tm值计算G+C含量，或由G+C含量计算Tm值。
绛旓細DNA鐔旇В娓╁害锛屾寚鎶奃NA鐨勫弻铻烘棆缁撴瀯闄嶈В涓鍗婃椂鐨勬俯搴︺備笉鍚屽簭鍒楃殑DNA锛孴m鍊间笉鍚屻侱NA涓璆锛岰鍚噺瓒婇珮锛孴m鍊艰秺楂橈紝鎴愭姣斿叧绯汇傛牳閰窽m鍊硷紙瑙ｉ摼娓╁害锛夎绠 A variety of factors affect the efficiency of hybridization between two strands of DNA. These include the nature of the hybridizing molecule...
绛旓細Tm鍊肩殑褰卞搷鍥犵礌 1銆丟鈥擟鐨勫惈閲忥細鍦ㄤ竴瀹氭潯浠朵笅Tm楂樹綆鐢盌NA鍒嗗瓙涓殑G-C鍚噺鎵鍐冲畾銆侴-C鍚噺楂樻椂锛孴m鍊兼瘮杈冮珮锛屽弽涔嬪垯浣庛傝繖鏄洜涓篏-C涔嬮棿鐨勬阿閿緝A-T澶氾紝瑙ｉ摼鏃堕渶瑕佽緝澶氱殑鑳介噺涔嬫晠銆俆m鍊煎拰G-C鐨勫惈閲忓彲鐢ㄤ簩涓涓粡楠屽叕寮忚〃绀猴細锛圙鈥擟锛%=锛圱m-69.3锛壝2.44銆傚湪涓瀹氭潯浠朵笅锛坧H7.0锛0.165...
绛旓細褰卞搷鍥犵礌锛欸+C姣斾緥瓒婇珮锛孴m鍊艰秺楂銆俆m=4锛圙+C锛+2锛圓+T锛Tm鍊间负PCR鍙嶅簲閫鐏俯搴︾殑閲嶈鍙傝冧緷鎹銆傞氬父灏嗗姞鐑彉鎬т娇DNA鐨勫弻铻烘棆缁撴瀯澶卞幓涓鍗婃椂鐨勬俯搴︾О涓鸿DNA鐨勭啍鐐规垨婧惰В娓╁害锛坢elting temperature锛夛紝鐢═m琛ㄧず銆傚綋杈惧埌Tm鏃讹紝DNA鍒嗗瓙鍐50%鐨勫弻閾剧粨鏋勮鎵撳紑锛屽嵆澧炶壊鏁堝簲杈惧埌涓鍗婃椂鐨勬俯搴︼紝瀹冨湪S鍨...
绛旓細G-C瀵瑰惈閲忥紝G-C涔嬮棿鐨勬阿閿緝A-T澶氥Tm鍊肩殑澶у皬涓嶥NA鍒嗗瓙涓⒈鍩虹殑缁勬垚銆佹瘮渚嬪叧绯诲拰DNA鍒嗗瓙鐨勯暱搴︽湁鍏銆傚湪DNA鍒嗗瓙涓紝濡傛灉G-C鍚噺杈冨锛孴m鍊煎垯杈冨ぇ锛孉-T鍚噺杈冨锛孴m鍊煎垯杈冨皬锛岃繖鏄洜涓篏-C涔嬮棿鐨勬阿閿緝A-T澶氾紝瑙ｉ摼鏃堕渶瑕佽緝澶氱殑鑳介噺涔嬫晠銆
绛旓細3.婧舵恫鐨凱H鍊銆傚湪楂榩H鍊间笅锛岀⒈琚箍娉涜劚璐ㄥ瓙锛屽け鍘诲舰鎴愭阿閿殑鑳藉姏銆傚綋pH鍊煎ぇ浜11.3鏃讹紝DNA瀹屽叏鍙樻с傚綋pH鍊煎皬浜5鏃讹紝DNA瀹规槗琚槍鍛ゅ寲銆傚綋鍗曢摼DNA鐢垫吵鏃讹紝灏嗘阿姘у寲閽犳坊鍔犲埌鍑濊兌涓互淇濇寔鍙樻с4.鍙樻у墏銆傜敳閰拌兒銆佸翱绱犲拰鐢查啗浼氱牬鍧忔阿閿紝闃荤纰辩Н绱紝闄嶄綆TM銆傝繖浜涘彉鎬у墏閫氬父鐢ㄤ簬鍗曢摼DNA鐨勭數娉炽俆m鍊...
绛旓細dna鍧囦竴鎬т笌tm鐨勫叧绯伙細DNA鐨勫垎瀛愬ぇ灏褰卞搷Tm鍊銆侱NA鐨勯暱搴﹁秺闀匡紝Tm鍊艰秺澶с傗憼 澶栭儴鍥犵礌锛歱H銆佺瀛愬己搴︺俻H杩囬珮鎴栬呰繃浣庨兘浼氶檷浣嶵m鍊笺傞殢鐫婧跺墏鍐呯瀛愬己搴︿笂鍗囷紝Tm鍊间篃闅忕潃澧炲ぇ銆傗憽鍐呴儴鍥犵礌锛欴NA鐨勭⒈鍩烘瘮渚嬨丏NA鐨勫潎涓鎬э紱鍦ㄧ浉鍚屾潯浠朵笅锛孌NA鍐匞-C閰嶅鍚噺楂橈紝鍏禩m鍊间篃楂樸侱NA瓒婂潎涓锛孴m鍊艰寖鍥磋秺绐...
绛旓細鍦―NA鍙岄摼鏉備氦杩囩▼涓紝澶氫釜鍥犵礌褰卞搷鍏舵晥鐜囷紝鍖呮嫭鏉傚悎鍒嗗瓙鐨勬ц川锛圖NA鎴朢NA锛夈侀暱搴︺佹潅鍚堢幆澧冿紙鐩愭祿搴﹀拰鍙樻у墏锛夈佹帰閽堟祿搴︿互鍙婂畠浠殑搴忓垪銆傚浜庤啘缁撳悎鐩爣鍜屼腑绛夐暱搴︾殑DNA鎺㈤拡锛孒owley绛変汉鍙戠幇锛50%鎺㈤拡涓庡叾浜掕ˉ閾剧粨鍚堢殑鐔旇В娓╁害锛Tm锛夊彲浠ラ氳繃浠ヤ笅鍏紡瀹氫箟锛歍m = 81.5 + 16.6logM + 41(%G + %C) ...
绛旓細2. DNA鐨"tm"鍊硷細"tm"鍊艰〃绀篋NA鐨勭啍瑙ｆ俯搴︼紝鍗冲湪浣曠娓╁害涓婦NA鐨勪袱鏉￠摼寮濮嬭В寮锛屽け鍘诲弻閾剧粨鏋勩傚湪杩欎釜杩囩▼涓紝姘㈤敭鏂锛屼娇涓ゆ潯閾惧垎绂伙紝褰㈡垚涓や釜鍗曢摼銆"tm"鍊奸氬父浠ユ憚姘忓害锛埪癈锛変负鍗曚綅琛ㄧず銆3. 褰卞搷"tm"鍊肩殑鍥犵礌锛"tm"鍊煎彈鍒板绉鍥犵礌鐨勫奖鍝锛屽寘鎷互涓嬪嚑涓噸瑕佸洜绱狅細DNA搴忓垪锛欴NA鐨勭⒈鍩哄簭鍒...
绛旓細褰卞搷Tm鍊肩殑鍥犵礌鏈夆︹ 閫塀,GC闂存湁涓変釜姘㈤敭,AT闂翠袱涓,鎵浠,GC鍚噺瓒婇珮,TM鍊艰秺澶с傜敓鐗╁寲瀛︾殑Tm 琛ㄧず鐨勬槸浠涔堚︹ 鐢熷寲閲岀殑Tm涓鑸〃绀篋NA鐔旇В娓╁害,鎸囨妸DNA鐨勫弻铻烘棆缁撴瀯闄嶈В涓鍗婃椂鐨勬俯搴︺備笉鍚屽簭鍒楃殑DNA,Tm鍊...鏍搁吀涓奖鍝峊m鍊肩殑鍥犵礌鏈夊摢浜?鈥︹ DNA鐔旇В娓╁害,鎸囨妸DNA鐨勫弻铻烘棆缁撴瀯闄嶈В涓鍗婃椂鐨...
绛旓細Tm鍊肩殑璁＄畻鏄熀浜嶥NA搴忓垪鐨鏍搁吀鐗╃悊鎬ц川锛屽寘鎷珼NA搴忓垪鐨勯暱搴︺佺⒈鍩哄簭鍒椼佹憾娑叉潯浠剁瓑銆俆m鍊肩殑璁＄畻鏂规硶鏈夊绉嶏紝鍏朵腑鏈甯哥敤鐨勬柟娉曟槸閫氳繃鍏紡璁＄畻锛屽叕寮忓寘鎷俯搴︺丯a+绂诲瓙娴撳害銆丏NA娴撳害鍜岀⒈鍩虹粍鎴愮瓑鍥犵礌銆傛澶栵紝杩樻湁璁稿鍦ㄧ嚎璁＄畻鍣ㄥ彲浠ュ揩閫熷噯纭湴璁＄畻Tm鍊笺俆m鍊煎湪瀹為獙瀹や腑鏈夌潃骞挎硾鐨勫簲鐢紝鍏朵腑鏈涓昏鐨勫簲鐢ㄦ槸鍦...
