title: Enhanced detection of DNA sequences using end-point PCR amplification and online gel electrophoresis (GE)-ICP-MS: determination of gene copy number variations.
abstract: The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ∼6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin.
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, ‡Department of Functional Biology (Genetic Area) and Oncology University Institute (IUOPA), University of Oviedo , C/Julian Clavería 8, 33006 Oviedo, Spain.
authors.forename: T Iglesias
authors.initials: TI
authors.lastname: González
authors.forename: M
authors.initials: M
authors.lastname: Espina
authors.forename: L M
authors.initials: LM
authors.lastname: Sierra
authors.forename: J
authors.initials: J
authors.lastname: Bettmer
authors.forename: E
authors.initials: E
authors.lastname: Blanco-González
authors.forename: M
authors.initials: M
authors.lastname: Montes-Bayón
authors.forename: A
authors.initials: A
authors.lastname: Sanz-Medel
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D056915:DNA Copy Number Variations; D004586:Electrophoresis; D006801:Humans; D020407:Internet; D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction / Q000295:instrumentation
chemical_list: D004247:DNA