Method for improving genetic stability of foreign insert nucleotide sequence in recombinant single-stranded RNA virus

A method for constructing a recombinant single-stranded RNA virus containing a foreign insert nucleotide sequence, which comprises the steps of:

• (a) preparing the foreign insert nucleotide sequence which has an even distribution of G/C content throughout the overall foreign insert nucleotide sequence and/or has a G/C content of more than 30%;
• and
• (b) introducing the foreign insert into a viral genome of a parent RNA virus to construct the recombinant RNA virus,
• wherein the foreign insert nucleotide sequence is introduced in such a manner that the the recombinant RNA virus is not disrupted for viral propagation

A method for amplifying a nucleotide sequence using template/ligation-free PCR method, which comprises the steps of:

• (a) preparing a plurality of DNA fragments serving as both template and primer, in which the DNA fragments are designed by dividing the entire nucleotide sequence of interest into several fragments with different complementary regions so that one segment is used as a template while being primed by the other;
• (b) mixing the DNA fragments in such as manner that the DNA fragments corresponding to both ends of final nucleotide sequence which is also used in amplification step has a higher concentration than the other DNA fragments;
• (c) preparing a full length of the nucleotide sequence of interest by PCR for 20-40 sec at 92-96° C. (denaturation), for 20-40 sec at 25-40° C. (annealing) and for 40-70 sec at 68-75° C. (extension);
• and
• (d) amplifying the nucleotide sequence of interest by PCR for 20-40 sec at 92-96° C. (denaturation) and 40-70 sec at 68-75° C. (annealing and extension).