Thermus aquaticus DNA连接酶的连接特性研究

Investigation of the Ligation Characteristics of Thermus aquaticus DNA Ligase

为进一步研究Thermus aquaticus（Taq）DNA连接酶的作用机理、开发以DNA连接作为关键步骤的新生物技术,本文以含有超稳定发卡结构的DNA（带有11nt长的单链部分）和另一条单链DNA为连接底物,研究了片段长度、反应温度、连接位点的结构特性、聚乙二醇（Polyethylene glycol,PEG）的添加等对连接的影响,探讨了Taq DNA连接酶的连接特性。连接结果显示：单链DNA的3’端同发卡结构I底物相连的连接率,比单链DNA的5’端同发卡结构II底物相连的连接率更高;可以连接的最短单链DNA片段的长度为6nt;在20～65℃范围内,一般连接率随温度升高而升高。在连接时,由2条底物通过互补配对形成的复合体的热稳定性对连接率影响不大,一般在连接温度远远高于该复合体的熔点（Melting Temperature,Tm）时仍有很高的连接率。在70或75℃时,虽然连接率有所降低,但仍然可以连接9nt的单链DNA片段。研究还发现,在片段连接上,PEG 6000对较难连接的片段（6～7nt）有促进作用,对容易连接的片段（8～10nt）促进作用不显著,甚至有一定抑制作用。

Background：DNA ligation is significant for both molecular biology and biotechnology.The ligation detail of T4 DNA ligase has been well studied and found that ligation can occur even when mismatched base pairs are present.Such scenario should be avoided when T4 DNA ligase is used for sequence specific DNA detection in which only positive result is expected for full matched case.On the other hand,the ligation characteristics of Thermus aquaticus（Taq）DNA ligase have not been studied in detail,especially for the temperature dependence.To further investigate the mechanism of Taq DNA ligase for the development of new biotechnologies using DNA ligation,here we used thermal stable DNA hairpin containing a 11 nt overhang and another single-stranded DNA as substrates to evaluate the effect of fragment length,temperature,structure of ligation sites,polyethylene glycol（PEG）concentration on DNA ligation by Taq DNA ligase.Results and Discussion：The results showed that structure of ligation substrates affected DNA ligation greatly.The yield of ligation between short DNA and hairpin I with 3＇overhang was much higher than that between hairpin II with 5＇overhang and short DNA with 5＇-phosphate.Interestingly,6nt short DNA could be ligated to both hairpin structures although the efficiency was not high.The difference in ligation efficiency may be caused by the differences of forming ligation intermediate attaching AMP.Usually,12 nt sticky ends are suggested to be used as ligation substrate,but we found 7or 8nt DNA could be ligated efficiently.This finding facilitated the molecular design of DNA detection technology.The ligation yield increased gradually with the increase of temperature from 20 to 65℃.Even for 8or 9nt short DNA which forms duplex（with its complementary sequence）with a Tmas low as 30℃,unexpectedly,the ligation efficiency was higher at 65℃than that at45 or 55℃.This demonstrated that the complex of DNA ligase and 5＇-phosphate substrate could help the hybridization.Even at 70 or 75℃,the 9nt single stranded DNA could still ligate to the substrate,although the efficiency became lower.It is obvious that the mismatched substrate should be difficult to be ligated.The results also revealed that PEG 6000 improved the ligation of short fragments（6～7nt）but not long ones（8～10nt）.Conclusions：Surprisingly,Taq DNA ligase could ligate a single-stranded DNA as short as 6nt.Even for DNA as short as 8～9nt,higher temperature（65 ℃）gave higher yield of ligation.Structure Hp-I was found to be a better substrate because it has 5＇phosphate,and could form the intermediate with Taq DNA ligase.These results are significant for developing DNA detection technology based on DNA ligation.