Patent ID: 7790420

Claim:
A method of generating a library of nucleic acid molecules of varying sizes, wherein each nucleic acid molecule forms an insert within a vector, and each vector comprises two pairs of restriction enzyme cleavage sites positioned so as to flank each side of the nucleic acid insert, the method comprising: a) cleaving a vector having a nucleic acid insert with a first pair of restriction enzymes which cleave at the pair of cleavage sites positioned in the vector on a flank of the nucleic acid insert such that an exonuclease-susceptible nucleic acid end results on an insert end of the vector and an exonuclease-resistant end results on a vector end; b) digesting the linearised vector using an exonuclease, followed by removal of single-stranded DNA; c) recircularising the vector by ligation of the exonuclease susceptible end and the exonuclease-resistant end; d) cleaving the recircularised vector with a second pair of restriction enzymes which cleave at the pair of cleavage sites positioned in the vector on a second flank of the nucleic acid insert, such that an exonuclease-susceptible nucleic acid end results on an insert end of the vector and an exonuclease-resistant end results on a vector end; e) digesting the linearised vector using an exonuclease, followed by removal of single-stranded DNA; and f) recircularising the vector by ligation; wherein step a) is performed on a plurality of vectors having the same insert, to produce the library of nucleic acid molecules comprises nucleic acid inserts truncated at both ends wherein the orientation of each nucleic acid insert within each vector is the same.