Microarray-based method for combinatorial library sequence mapping and characterization.
Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.
Abécassis
V
V
Centre National de la Recherche Scientifique, UPR 2167, Gifsur-Yvette, France.
Jaffrelo
L
L
Rickman
D
D
Aggerbeck
L
L
Herbert
C
C
Truan
G
G
Pompon
D
D
D001483:Base Sequence; D001709:Biotechnology; D002874:Chromosome Mapping / Q000379:methods; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000706:statistics & numerical data; D015345:Oligonucleotide Probes / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2