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為甚麼Taq可以耐高溫工作
為甚麼肉類要低溫保鮮 (引注資料 (https://www.ncbi.nlm.nih…
為甚麼Taq可以耐高溫工作
為甚麼肉類要低溫保鮮
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引注資料
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Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability
https://goo.gl/4ELvRd
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為何Taq可以耐高溫
結構
跟Ecoli同源性很高
三個domain:
– Polymerase Activity
– 3’ to 5’ Exonuclease - Fix errors
– 5’ to 3’ Exonuclease – Remove RNA or DNA in “front” of primer
The fact that there is significant similarity in native states but dissimilarity in stability, points to the involvement of denatured states.
The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities
Taq酶跟E.coli的pol同源,都有domain可以去做5'的活性
序列:
TP has more arginines instead of lysines, more glycines instead of alanines, more prolines instead of alanines, and a slight increase in hydrophobic residue content.
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在高熱度下面還是很穩定
Crystal structure of Taq has more stable interactions (or less unfavorable interactions) compared E. coli Pol I polymerase, nothing significant to explain thermostability
Folding enthalpy of Taq polymerase is less than that of E. coli Pol I polymerase. Taq is more stable due to reduced entropic penalty of folding
Many thermophilic proteins are stable at high temperatures due to enthalpic contributions. That is, more hydrogen bonds/salt bridges/non-covalent interactions
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本身活性
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使合成的產物「末端帶有一個A鹼基」,TA克隆即是利用Taq聚合酶此特性,Taq的PCR產物在3'末端會多出一個A,此時只須有一個與其互補的T表現在質體上,即能彼此靠近,藉由接合酶連接起來。透過此方式,可省去限制酶剪切的時間,直接利用PCR產物與質體彼此有互補兩端的特性,可快速黏合起來。
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