公开(公告)号：KR101488110B1

公开(公告)日：2015-01-29

申请号：KR1020130147812

申请日：2013-11-29

Applicant: 성균관대학교산학협력단

Inventor： 권석태 ,  조성숙 ,  유미 ,  권경민 ,  김승현

IPC: C12N9/12 ,  C12N15/54 ,  C12Q1/48 ,  C12Q1/68

CPC classification number: C12N9/1252 ,  C07K2319/00 ,  C07K2319/21 ,  C12Q1/686 ,  C12Q2521/101 ,  C12Q2549/101

Abstract: A DNA polymerase (Neq DNA polymerase) derived from Nanoarchaeum equitans is divided into a Neq DNA polymerase large fragment (NeqL) and a NeqDNA polymerase small fragment (NeqS), and the fragments have intein respectively. The present invention connects inteins of the NeqL fragment and the NeqS fragment into one and provides a Neq hot-start (HS) DNA polymerase in a form of a precursor of the Neq DNA polymerase. The Neq HS DNA polymerase has an advantage of reducing inconvenience of being used for hot-start PCR by an intein trans splicing by purifying the existing NeqL fragment and NeqS fragment respectively. Especially, six histidine (His-tag) sequence is inserted in the level of gene between the intein of the NeqL fragment and the intein of NeqS fragment to significantly improve the purifying method. Additionally, as a result of an effort for increasing PCR efficiency of Neq HS DNA polymerase, mutation is induced on a specific part of Neq HS DNA polymerase genes to select Neq HS mutant polymerases (M1, M2, and M3) having significantly improved PCR amplification rate and amplification amount.

Abstract translation: 将衍生自Nanoarchaeum equitans的DNA聚合酶（Neq DNA聚合酶）分为Neq DNA聚合酶大片段（NeqL）和NeqDNA聚合酶小片段（NeqS），分别具有内含肽。 本发明将NeqL片段和NeqS片段的内含肽连接在一起，并以Neq DNA聚合酶的前体形式提供Neq热启动（HS）DNA聚合酶。 Neq HS DNA聚合酶具有通过分别纯化现有的NeqL片段和NeqS片段，通过内含肽的剪接减少用于热启动PCR的不便的优点。 特别地，将六组氨酸（His-标签）序列插入NeqL片段的内含肽与NeqS片段的内含肽之间的基因水平，以显着提高纯化方法。 另外，作为提高Neq HS DNA聚合酶的PCR效率的努力的结果，在特定部分的Neq HS DNA聚合酶基因上诱导突变以选择具有显着改善的PCR扩增的Neq HS突变型聚合酶（M1，M2和M3） 速率和放大量。