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Enhanced Detection of DNA Sequences Using End-Point PCR Amplification and Online Gel Electrophoresis (GE)-ICP-MS: Determination of Gene Copy Number Variations
- Source :
- Analytical Chemistry. 86:11028-11032
- Publication Year :
- 2014
- Publisher :
- American Chemical Society (ACS), 2014.
-
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.
- Subjects :
- Electrophoresis
Gel electrophoresis
Internet
Base Sequence
DNA Copy Number Variations
DNA
Polymerase Chain Reaction
Molecular biology
Mass Spectrometry
DNA sequencing
Cell Line
Analytical Chemistry
law.invention
genomic DNA
chemistry.chemical_compound
Real-time polymerase chain reaction
chemistry
law
Agarose gel electrophoresis
Humans
Copy-number variation
Polymerase chain reaction
Subjects
Details
- ISSN :
- 15206882 and 00032700
- Volume :
- 86
- Database :
- OpenAIRE
- Journal :
- Analytical Chemistry
- Accession number :
- edsair.doi.dedup.....89bc4ec14060c38dd7f01c918603a11a