title: Analysis of recombinant adenoviruses using an integrated microfluidic chip-based system.
abstract: The use of recombinant virus for gene therapy requires rigorous quality control methods to ensure that the viral vector preparations are functional and safe. A viral identity test is performed in which the viral payload, or transgene, is PCR amplified, followed by digestion with restriction enzymes that yields a characteristic "fingerprint." These DNA fragments are typically analyzed by agarose gel electrophoresis. The ethidium bromide-stained gels are photographed or scanned and the results are sufficient for a qualitative or semiquantitative identity confirmation of the viral product. We have investigated the use of an integrated microfluidic chip-based system as a new tool in the quality control testing of a recombinant, adenoviral, gene therapy product. The chip-based method was found to be very sensitive, requiring 100-fold less sample and only one-third the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.
authors.affiliation: Berlex Laboratories, Inc., Richmond, California 94804, USA. mike_mccaman@berlex.com
authors.forename: M T
authors.initials: MT
authors.lastname: McCaman
authors.forename: P
authors.initials: P
authors.lastname: Murakami
authors.forename: E
authors.initials: E
authors.lastname: Pungor
authors.forename: K M
authors.initials: KM
authors.lastname: Hahnenberger
authors.forename: W S
authors.initials: WS
authors.lastname: Hancock
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics*; D002460:Cell Line; D016172:DNA Fingerprinting / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry; D004586:Electrophoresis; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D005898:Glass; D006801:Humans; D008970:Molecular Weight; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004274:DNA, Recombinant; D004279:DNA, Viral