Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor
Introduction
It has been estimated that 60% of all humans have been affected by gene mutation in their lifetime. This, rather dramatically, demonstrates the importance of increasing demand for new high-throughput methods for mutation detection.
A variety of methods are currently used to assess the mutation status of individual tumours (Nollau and Wagener, 1997). TP53 gene is mutated in most types of human cancers and is one of the most studied genes in cancer research. Moreover, many studies have suggested that TP53 mutations have prognostic importance and sometimes are determinant in the response of tumours to therapy. The role of TP53 as an important early diagnostic marker and its mutation spectrum have emerged and traditional methods for detecting point mutations as well as new approaches based on biosensors and DNA chips for detecting and recognizing mutations of TP53 have been developed. This has been reviewed very recently by Jiang et al. (2004).
Among traditional methods for molecular diagnosis and also for TP53, distinction is made between point mutation scanning and screening technologies. Scanning technologies aim at finding unknown mutations in candidate or known disease genes, such as direct DNA sequencing. Screening techniques aim at finding known mutations, preferably with high throughput (Jiang et al., 2004). For example, denaturing high-performance liquid chromatography (DHPLC) (Narayanaswami and Taylor, 2002, Gross et al., 2001), Single-strand conformation polymorphism (SSCP) (Miyajima et al., 2001, Ru et al., 2000), and denaturing gradient gel electrophoresis (DGGE) (Sheffield et al., 1989, Van Orsouw et al., 1998). However, some of these approaches are time consuming and require highly skilled labor, while some are less sensitive or use expensive equipment.
In recent years, a new trend for the detection of TP53 mutations has aimed at label-free, high-sensitivity and specificity, real-time and rapid detection. Biosensors, in particular DNA-based sensors, and gene chips are of considerable recent interest due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional hybridisation assays. Different transduction principles have been employed for TP53 DNA detection including electrochemical, piezoelectric and optical (SPR) techniques. All these systems are based on the hybridisation reaction between a probe immobilised on the transducer surface, which may be either an electrode, in particular a carbon paste (Palacĕk et al., 1998) or a gold electrode (Wang et al., 1997a, Miyahara et al., 2002), a quartz crystal coated with gold (Wang et al., 1997b, Wittung-Shafshede et al., 2000) or a sensor chip (Nilsson et al., 1997, Nilsson et al., 1999) made of glass with evaporated gold on one side in the case of SPR sensing. Most of the papers relating to TP53 detection in the literature use only standard solutions containing synthetic oligonucleotides, respectively, complementary and containing a mutation (mismatch: point mutation in one base). Only Miyahara et al. and Nilsson et al. deal with real samples consisting of PCR-amplified DNA from blood or tissues, in particular, from microdissected tumor biopsies, containing the target sequence able to hybridise with the immobilised probe.
In the current work, we report a method for detecting TP53 mutations using a new portable surface plasmon resonance-based biosensor. In particular, we employed the inexpensive, portable and commercially available instrument, SPREETA™ SPR. The system is based on the hybridisation reaction between the immobilised probe and its complementary or mismatched sequence in solution. The probe immobilisation is based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces. This procedure has been successfully employed with SPR DNA-based sensing in previous work by our group using both the SpreetaTM and Biacore X™ instruments (Wang et al., 2004a, Wang et al., 2004b). The system was optimized using synthetic oligonucleotides and the main analytical parameters of the sensor (selectivity, sensitivity, reproducibility, analysis time etc.) were studied in detail. The system was applied to complementary and a mismatch sequences (C → A) corresponding to codon 248 of the TP53 gene. DNA extracted from “normal” wild-type cell line (Jurkat) containing the fully complementary sequence and DNA extracted from cell line (Molt 4) carrying that mutation was also tested prior to amplification by PCR.
Section snippets
Apparatus and reagents
For all the experiments the SPR device Spreeta™ (Texas Instruments Inc., USA) and a bare gold Spreeta™ sensor were used. All experiments were conducted at a flow rate of 5 μl/min and 25 °C.
6-Mercapto-1-hexanol (MCH) was purchased from Sigma–Aldrich (Milan, Italy). Other reagents for the buffers were purchased from Merck (Darmstadt, Germany). The composition of the buffers used for the experiments is as follows:
immobilisation solution: KH2PO4 1 M, pH 3.8;
hybridisation buffer: NaCl 150 mM, Na2HPO4 20
Results and discussion
Preliminary experiments were conducted with the 25-mer oligonucleotides to verify the ability of the sensor to generate a specific and reproducible signal when in contact with the fully complementary sequence solution. Initially, the probe was immobilised on the sensor surface and the operating conditions optimized.
Conclusions
We have successfully applied a portable surface plasmon resonance (SPR) biosensor to point mutation detection. The system is based on hybridisation detection between a DNA probe immobilised on the sensor surface and the target sequence in solution. The commercially available instrument SPREETA™ SPR-EVM-BT was used for the analysis of PCR-amplified samples to detect TP53 mutation.
Probe immobilisation was achieved by direct coupling of thiolated probes on gold sensor surfaces. The SPR-based
Acknowledgements
We thank Dr. Lucia Mosiello, Enea-Casaccia, Italy, for cultivating Jurkat and Molt 4 cell lines and for DNA extraction. One of the authors (Maria Minunni) would like to thank the Italian Ministery of Health (Ministero della Sanità) for financial support: Project “Inibitori della ciclossigenasi-2 (COX2) nel trattamento dei tumori colorettali” for financial support.
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