Most studies of p53 have defined mutations by using immunohistochemistry (IHC), even if the use of IHC as an indirect measure of p53 status gives ambiguous results.
Later, several molecular methods are developed for rapid prescreening of p53 mutations, which do not give any information on the nature of the mutation. They can be grouped as either conformation-based techniques or base mismatch recognition techniques (for review, see Ref. ).
The conformation-based procedures for mutation detection are single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), denaturing gradient gel electrophoresis (DGGE), and modifications of DGGE: temperature gradient gel electrophoresis (TGGE) and constant gradient gel electrophoresis (CDGE). These techniques utilize the property that DNA fragments containing a sequence alteration have altered mobility under certain conditions of gel electrophoresis compared with a control.
Base mismatch recognition methods exploit the ability of chemicals or proteins (e.g., endonuclease V, RNase, or MutS and MutY mismatch repair proteins) to recognize mismatched bases in DNA heteroduplexes. The singlestrand specificity of RNase has similarly been utilized to digest RNA:DNA or RNA:RNA (nonisotopic RNase cleavage assay or NIRCA) heteroduplex.
In recent years, the previously described technologies have been significantly simplified and automated to make them less time-consuming, and they have been coupled together, producing a variety of methodological approaches for p53 mutation detection. The possibility of binding heteroduplex and oligonucleotides arrays to solid and semisolid supports has led to automation. Moreover, the possibility to detect DNA fragments of different sizes by capillary electrophoresis (CE) instead of laborintensive gel electrophoresis, miniaturization (chip technology), as well as the use of fluorophores instead of dangerous reagents have all drastically reduced the drawbacks of the above methods and have improved their sensitivity (100%) and throughput.
Moreover, the increased efficiency of DNA sequencing analysis is an important goal in modern biology. Direct sequence analysis is the most precise method for determining p53 mutation status (100% specificity). Automated sequencing has streamlined sequence data production and acquisition.
Another recent approach, pyrosequencing, has shown accurate results for p53 mutation detection. Pyrosequenc-ing is a nonelectrophoretic real-time DNA sequencing method that uses luciferase-luciferin light release as the detection signal for nucleotide incorporation into a target DNA.
Advances in genomics, proteomics, bioinformatics, and nanotechnology have recently increased exponentially. The integration of these scientific fields with multiplexed assays allows the development of devices that undoubtedly enable higher throughput both in screening of mutations and sequencing determination: the chip technology.
The high-density oligonucleotide arrays (DNA chip), attached to a solid glass or nylon matrix, allow the rapid scanning of any gene to determine the zygosity (i.e., discrimination between homozygotes and heterozygotes) for all possible allelic variations in patient samples (mutations and polymorphisms) and expression studies. Dedicated instrumentation and software allow fluorescence detection, data acquisition, and analysis of hybridization patterns.
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