Automated two-column purification of iminobiotin and BrdU-labeled PCR products for rapid cloning: application to genes synthesized by polymerase chain assembly.
Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made in de novo gene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full-length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the pUC19 beta-lactamase gene (929 bp). Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.
TerMaat
Joel R
JR
Chemical & Biomolecular Engineering, 820 N. 16th St, University of Nebraska-Lincoln, Lincoln, NE 68588-0643, United States. jtermaat@huskers.unl.edu
Mamedov
Tarlan G
TG
Pienaar
Elsje
E
Whitney
Scott E
SE
Subramanian
Anuradha
A
D000345:Affinity Labels / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D001973:Bromodeoxyuridine / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005796:Genes; D016133:Polymerase Chain Reaction; D001618:beta-Lactamases / Q000235:genetics
D000345:Affinity Labels; C518163:iminobiotin; D001710:Biotin; D004247:DNA; C096780:beta-lactamase TEM-pUC19; D001618:beta-Lactamases; D001973:Bromodeoxyuridine