Compositions, kits, and methods for analysis of dna sequence-specificity in v(d)j recombination

A high throughput method of analyzing DNA sequence-specificity in a V(D)J recombination assay, the method comprising the steps of:

• producing a plasmid library comprising a plurality of plasmid constructs, each plasmid construct comprising a plasmid vector having a 12-recombination signal sequence (12-RSS) and a 23-recombination signal sequence (23-RSS) inserted therein in a colinear orientation and a segment of at least 100 base pairs inserted in between the 12-RSS and the 23-RSS, wherein the 12-RSS comprises a heptamer beginning with a sequence of CAC, a 12 base pair spacer, and a nonamer, and wherein the 23-RSS comprises a heptamer beginning with a sequence of CAC, a 23 base pair spacer, and a nonamer, wherein at least one of the 12-RSS and the 23-RSS in each plasmid construct has a degenerate base pair sequence of one to ten consecutive base pairs present therein that does not include the CAC sequence of the heptamers, and wherein the plasmid library comprises about 4n plasmids, wherein n is the number of base pairs present in the degenerate base pair sequence, and wherein the method of producing the plasmid library comprises the steps of:
• producing a first plurality of synthetic oligonucleotides that comprise the 12-RSS having the degenerate base pair sequence of one to ten consecutive base pairs present therein, wherein the first plurality of synthetic oligonucleotides comprises about 4n oligonucleotides, wherein n is the number of base pairs present in the degenerate base pair sequence;
• producing a second plurality of synthetic oligonucleotides that comprise the 23-RSS having the degenerate base pair sequence of one to ten consecutive base pairs present therein, wherein the second plurality of synthetic oligonucleotides comprises about 4n oligonucleotides, wherein n is the number of base pairs present in the degenerate base pair sequence;
• converting the first and second pluralities of synthetic oligonucleotides to double-stranded DNA;
• linearizing a plasmid, wherein the plasmid comprises canonical 12-RSS and canonical 23-RSS present therein in a colinear orientation and the segment of at least 100 base pairs disposed therebetween;
• removing the canonical 12-RSS and 23-RSS;
• and
• ligating the double-stranded DNA comprising the first and second pluralities of synthetic oligonucleotides to the plasmid to produce the plasmid library;
• transfecting mammalian cells with the plasmid library, wherein the mammalian cells are capable of expressing recombination activating gene proteins 1 and 2 (RAG1 and RAG2);
• culturing the transfected cells under conditions that allow for expression of RAG1 and RAG2 and production of recombination products in which the portion of the plasmids between the 12-RSS and 23-RSS is inverted to form a 12-RSS:23-RSS signal joint and a coding joint;
• harvesting the transfected mammalian cells;
• recovering plasmid DNA from the harvested cells;
• and
• selectively amplifying the recombination products using primers that amplify the 12-RSS:23-RSS signal joint formed during recombination, wherein the selectively amplified recombination products constitute an output library for the recombination assay.