Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis.
With the increasing availability of genetic information and its relationship to human diseases, there is a growing need in the medical diagnostic field for technologies that can proceed to the parallel genotyping of multiple markers. In this paper, we report the development of a new flexible microarray-based method that aims to be inexpensive, accurate, and adapted to routine analysis. The construction of the MICAM (MICrosystem for Analysis in Medicine) DNA chip is based on the controlled electro-synthesis of a conducting polymer film bearing oligonucleotide probes on gold electrodes. First, accessible 3'OH-ends of grafted probes are directly used to conduct single template-dependent nucleotide extension reactions with fluorescence-labeled chain terminators. Then, the fluorescence of incorporated dideoxynucleotides on controls and probes of interest are recorded to assess base calling. Here, we present the development of the methodology to assign the genotype of TP53 (tumor protein p53) codon 72 polymorphism and its application to analysis of genomic DNA from cell lines and from human colorectal samples. The genotyping results obtained by mini-sequencing on the polypyrrole DNA chip were 100% concordant with data obtained by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Moreover, the developed probe array assay has been successfully applied to the detection of TP53 loss of heterozygosity.
Ho-Pun-Cheung
Alexandre
A
Cancer Research Center, Val d'Aurelle Cancer Institute, Montpellier, France.
Choblet
Sylvie
S
Colineau
Thomas
T
Abaibou
Hafid
H
Zsoldos
Daniela
D
Brengel-Pesce
Karen
K
Grenier
Jean
J
Cleuziat
Philippe
P
Lopez-Crapez
Evelyne
E
D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis*; D019937:Diagnostic Techniques and Procedures; D020869:Gene Expression Profiling; D005838:Genotype; D006801:Humans; D019656:Loss of Heterozygosity; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D004273:DNA, Neoplasm; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53