Abstract: A method for detecting target nucleic acids in a sample is disclosed. The method can rapidly detect target nucleic acids and has simplified protocols. Therefore, the method can be effectively used by large hospitals, entrusted testing institutions, research laboratories, and the like at which rapid molecular diagnosis for a large number of samples is required.

Abstract: A DNA polymerase (Neq DNA polymerase) derived from Nanoarchaeum equitans is split into Neq L and Neq S fragments, each of which contains inteins. A Neq hot-start (HS) DNA polymerase in which the inteins of the Neq L and Neq S fragments are linked with each other is provided in the form of a precursor of Neq DNA polymerase. A purification method can be significantly improved by inserting a His-tag sequence composed of six histidine residues between the inteins of the Neq L and Neq S fragments at a gene level. As a result of effort to enhance PCR efficiency of the Neq HS DNA polymerase, a gene coding for the Neq HS DNA polymerase is mutated at specific positions to screen mutant Neq HS polymerases (M1, M2, and M3) having a highly improved PCR amplification rate and amplification level.

Abstract: A DNA polymerase (Neq DNA polymerase) derived from Nanoarchaeum equitans is split into Neq L and Neq S fragments, each of which contains inteins. A Neq hot-start (HS) DNA polymerase in which the inteins of the Neq L and Neq S fragments are linked with each other is provided in the form of a precursor of Neq DNA polymerase. A purification method can be significantly improved by inserting a His-tag sequence composed of six histidine residues between the inteins of the Neq L and Neq S fragments at a gene level. As a result of effort to enhance PCR efficiency of the Neq HS DNA polymerase, a gene coding for the Neq HS DNA polymerase is mutated at specific positions to screen mutant Neq HS polymerases (M1, M2, and M3) having a highly improved PCR amplification rate and amplification level.

Abstract: Disclosed is a hot-start PCR method, based on protein trans-splicing of intein-inserted large (Neq L) and small (Neq S) fragments of Neq DNA polymerase. The method comprises: preparing a PCR reaction mixture containing a sample DNA and primers; adding the Neq L fragment and the Neq S fragment together to the PCR reaction mixture, said Neq L fragment consisting of an amino acid sequence of SEQ ID NO: 2, with an intein amino acid sequence stretching from position 579 to 676 therein, said Neq S fragment consisting of an amino acid sequence of SEQ ID NO: 4 with an intein amino acid sequence stretching from position 1 to 30 therein; inducing the Neq L fragment and the Neq S fragment to undergo a protein trans-splicing process to form a polypeptide exhibiting Neq DNA polymerase activity; and performing a certain number of cycles of DNA denaturation, primer annealing and DNA extension.

Abstract: Disclosed herein is a mutant Neq A523R DNA polymerase consisting of an amino acid sequence of SEQ ID NO: 2 wherein alanine at amino acid position 523 in a Nanoarchaeum equitans DNA polymerase (Neq DNA polymerase) consisting of an amino acid sequence of SEQ ID NO: 1 has been substituted with arginine by site-directed mutagenesis. Also disclosed are a gene consisting of a nucleotide sequence encoding the mutant Neq A523R DNA polymerase, a recombinant vector comprising the gene, and a transformant transformed with the vector. In addition, disclosed are a PCR kit comprising the mutant Neq A523R DNA polymerase having excellent performance compared to the wild-type Neq DNA polymerase, and a method for preparing the Neq A523R DNA polymerase.