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Enhanced detection of DNA sequences using end-point PCR amplification and on-line gel electrophoresis (GE)ICP-MS: determination of gene copy number variations Tamara Iglesias, Marta Espina, Luisa Maria Sierra, Joerg Bettmer, Elisa Blanco Gonzalez, Maria Montes-Bayon, and Alfredo Sanz-Medel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac502671f • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 19, 2014
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Analytical Chemistry
Enhanced detection of DNA sequences using end-point PCR amplification and on-line gel electrophoresis (GE)-ICP-MS: determination of gene copy number variations T. Iglesias González,1 M. Espina,2 L. M. Sierra,2 J. Bettmer,1 E. Blanco-González1*, M. Montes-Bayón1* and A. Sanz-Medel1
1
Department of Physical and Analytical Chemistry. Faculty of Chemistry. University of
Oviedo. C/ Julian Clavería 8, 33006 Oviedo. Spain. 2
Department of Functional Biology (Genetic Area) and Oncology University Institute
(IUOPA). University of Oviedo.
[email protected] [email protected] ACS Paragon Plus Environment
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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 labelling 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 detection of phosphorous in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits the 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 P (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 relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit in terms of required amount of starting DNA is approximately 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 like cisplatin.
Key-words: Amplification, genes, gel electrophoresis, inductively coupled plasma mass spectrometry, quantification.
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Analytical Chemistry
Introduction Changes in copy number of genes contribute to the pathogenesis of various genetic disorders and cancer.1 Copy number variations (CNVs) are imbalances that alter the diploid status of a locus so that copy numbers increase (duplications) or decrease (deletions). 2 The screening of genome-wide CNVs in single cells is of special importance for a variety of applications in basic research and clinical diagnosis of diseases like Alzheimer's disease, autism, schizophrenia, breast cancer or obesity.3,4 In addition, recent studies have associated genomic CNVs with chemoresistance (tumoral cells that are intrinsically resistant to chemotherapy or become resistant after an initial partial response).5,6 Therefore, the development of suitable technologies for wholegenome amplification protocols (particularly in single cells) and appropriate platforms for evaluating the amplification products is highly demanded. The first generation of PCR users performed end-point analysis by gel electrophoresis and added fluorogenic intercalating dyes like ethidium bromide or SYBR® Green for signal monitoring.7 The introduction of real-time (or so-called quantitative) PCR (qPCR) spawned a second generation of assays that enabled quantitation by monitoring the progression of the amplification after each cycle using fluorescence probes. 8 , 9 In real-time PCR, quantitative information is obtained from the cycle threshold (CT), a point on the analogue fluorescence curve where the signal increases above background.10 External calibrators or normalization to endogenous controls are required to estimate the concentration of an unknown sample. When relative quantification is not sufficient and absolute numbers are required it is necessary to sort to quantification standards (RNA, cDNA, plasmid DNA, genomic DNA) and the accuracy of the result largely depend on the accuracy of the used standard. Accurate estimation therefore necessitates that
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the same reaction conditions are used for a standard and for the sample.11,12 In general, qPCR assays are characterized by a wide dynamic range of quantification of 7–8 logarithmic decades, a high technical sensitivity (
