Patent ID: 8293503
Filing Date: 2012-10-23
Classification: C12N,G16B

Abstract:
1. A method for the directional subcloning of DNA fragments comprising: a) providing a first vector comprising a first selectable marker gene and a DNA sequence of interest having an open reading frame, which DNA sequence of interest is flanked by at least two restriction enzyme sites, wherein one of the flanking restriction enzyme sites is a site for a first restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates complementary single-strand DNA 3′ TA overhangs, wherein the other flanking restriction enzyme site is for a second restriction enzyme which has infrequent restriction sites in cDNAs or open reading frames from at least one species and generates blunt ends, wherein digestion of the first vector with the first restriction enzyme and the second restriction enzyme generates: b) providing a second vector comprising a second selectable marker gene which is distinguishable from the first selectable marker gene, and a DNA sequence encoding a lethal gene flanked by at least two restriction enzymes sites, wherein one of the flanking restriction enzyme sites in the second vector is for a third restriction enzyme which generates complementary single-strand DNA overhangs that are complementary to the single-strand DNA overhang generated by the first restriction enzyme in the first linear DNA fragment, wherein the other flanking restriction site in the second vector is for a fourth restriction enzyme which generates blunt ends, wherein digestion of the second vector with the third restriction enzyme and the fourth restriction enzyme generates: wherein sequences including a portion of the overhang generated by the third restriction enzyme include one or more codons that after oriented joining are in-frame with the open reading frame, thereby encoding a N-terminal fusion with the gene product encoded by the open reading frame, and/or wherein sequences forming a portion of and 3′ to the cleavage site generated by the fourth restriction enzyme include one or more nucleotides (I) for a stop codon that after oriented joining are in-frame with the open reading frame or (II) for one or more codons that after oriented joining are in-frame with the open reading frame and thereby encode a C-terminal fusion with the gene product encoded by the open reading frame; c) combining in a suitable buffer with one or more restriction enzymes and DNA ligase under conditions effective to result in digestion and ligation to yield a mixture comprising a third vector comprising the first and third linear DNA molecules joined in an oriented manner.