Hot-start pcr based on the protein trans-splicing of nanoarchaeum equitans dna polymerase

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, comprising:

• preparing a PCR reaction mixture containing a sample DNA and primers;
• adding a Neq L fragment and a Neq S fragment together to the PCR reaction mixture, said Neq L fragment comprising an amino acid sequence having about 95% to less than 100% similarity to SEQ ID NO:2, with an intein amino acid sequence stretching from position 579 to 676 of SEQ ID NO:2, said Neq S fragment comprising an amino acid sequence having about 95% to less than 100% similarity to SEQ ID NO:
• 4 with an intein amino acid sequence stretching from position 1 to 30 of SEQ ID NO:
• 4;
• inducing the Neq L fragment and the Neq S fragment to undergo a protein trans-splicing process to form a polypeptide comprising an amino acid sequence having about 95% to less than 100% similarity to SEQ ID NO:
• 5 exhibiting Neq DNA polymerase activity;
• and
• performing a certain number of cycles of DNA denaturation, primer annealing and DNA extension.