Patent ID: 7279278

Claim:
A method for orientation-directed construction of a construct comprising at least two nucleic acid segments of interest, which method comprises the steps of: a. providing products having phosphorylated blunt ends derived from said nucleic acid segments of interest; b. performing separate ligation reactions, wherein in each reaction the phosphorylated blunt-ended products of two different segments obtained in step (a) are ligated to create a combined ligated sequence; c. performing PCR amplification reaction using said combined ligated sequence obtained in step (b) as a template and specific enrichment primers directing said PCR amplification towards the desired orientation of the complete combined ligated sequences, wherein each combined ligated sequence is amplified in a separate reaction creating a combined product having phosphorylated blunt ends; d. isolating and purifying said combined product having the enriched desired orientation obtained in step (c); and e. performing self-ligation of said isolated purified combined product obtained in step (d), creating a circular double-stranded DNA construct containing the desired segments properly aligned and operably linked.