RESUMEN
Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1-3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from approximately 60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of approximately 1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.
Asunto(s)
Barajamiento de ADN/métodos , Genes Sintéticos , Oligodesoxirribonucleótidos/síntesis química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/genética , Modelos Teóricos , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutación , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.