Non-random method of gene shuffling

A method for assembling DNA molecules in a non-random order in a DNA construct by

• (a) providing at least two double stranded template DNA molecules encoding members of a gene family and possessing regions of variation and of conservation along their DNA sequence;
• (b) designing oligonucleotide primers based on conserved sequences between each of the template molecules, wherein the primers also allow for the generation of single stranded 3′ or 5′ nucleic acid tails on an amplified nucleic acid product produced using these primers;
• (c) amplifying complementary nucleic acid products of each template DNA molecule using the designed oligonucleotide primers and allowing the complementary nucleic acid products to anneal together to form substantially double stranded nucleic acid molecules;
• (d) identifying or creating single stranded 3′ or 5′ single stranded terminal tails on the double stranded nucleic acid molecules, wherein the terminal single stranded nucleic acid tails have a length of from 2 to 30 nucleotides, wherein terminal single-stranded nucleic acid tails on a single double-stranded nucleic acid molecule do not hybridize to each other, wherein a terminal single-stranded nucleic acid tail on a double-stranded nucleic acid molecule is capable of hybridizing to a terminal single-stranded nucleic acid tail extending from a different double-stranded nucleic acid molecule or to a single-stranded DNA oligomer of from about 2 to about 30 nucleotides to allow for assembly of the nucleic molecules in a non-random order;
• and
• (e) incubating said nucleic acid molecules under conditions suitable to promote the assembling of the molecules in a non-random order to create a nucleic acid construct;
• wherein there are 2 or more possible orders for the assembly of the nucleic acid molecules.