title: Assembly of highly diverse genes using degenerate oligonucleotides by temperature cascade.
abstract: The quality of a nucleotide-based library such as a synthetic antibody library is highly dependent on the diversity available. Diversity can be generated using degenerate oligonucleotides introduced during gene assembly. Conventional approaches to gene assembly are not efficient for oligonucleotides with long stretches of degeneracy. We propose an efficient alternative for simultaneous introduction of three randomized regions in a synthetic antibody gene via temperature cascading. The strategy takes advantage of DNA reannealing kinetics. The strategy can be adopted for generating diversity of gene inserts during the construction of nucleotide-based libraries.
authors.affiliation: Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
authors.forename: Qiuting
authors.initials: Q
authors.lastname: Loh
authors.forename: Nur Hidayah Hairul
authors.initials: NH
authors.lastname: Bahara
authors.forename: Yee Siew
authors.initials: YS
authors.lastname: Choong
authors.forename: Theam Soon
authors.initials: TS
authors.lastname: Lim
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D000906:Antibodies; D009841:Oligonucleotides