Primer-extension based method for the generation of siRNA/miRNA expression vectors

A method for making an shRNA expression vector, comprising the steps of

• a. providing first and second DNA oligonucleotide primers, wherein
• i) said first DNA oligonucleotide primer comprises sequences encoding
• at a 3′ end, a first RNA loop sequence;
• at a 5′ end, a first restriction enzyme recognition sequence;
• and
• a first RNA stem sequence, wherein in said first DNA oligonucleotide primer, said first RNA stem sequence is located between said first RNA loop sequence and said first restriction enzyme recognition sequence;
• and
• ii) said second DNA oligonucleotide primer comprises sequences encoding
• a second RNA loop sequence that is complementary to said first RNA loop sequence;
• at a 3′ end, a second restriction enzyme recognition sequence;
• and
• at a 5′ end, a second RNA stem sequence, wherein said second RNA stem sequence is located between said second RNA loop sequence and said second restriction enzyme recognition sequence in said second DNA oligonucleotide primer;
• b. annealing said first RNA loop sequence and said RNA loop sequence to form an annealed structure;
• c. filling in single strand segments of said annealed structure to form a double-strand structure;
• d. cleaving said double-strand structure using restriction enzymes that recognize said first and said second restriction enzyme recognition sequences, to form a structure with a 3′ overhang and a 5′ overhang;
• and,
• e. ligating said structure with a 3′ overhang and a 5′ overhang into an expression vector to form an shRNA expression vector;
• and wherein said first RNA loop sequence and said second RNA loop sequence are 8-10 Nucleotides in length.