Methodology article

Low density DNA microarray for detection of most frequent TP53 missense point mutations

Angélica Rangel-López^1,2 , Rogelio Maldonado-Rodríguez¹ , Mauricio Salcedo-Vargas² , Juana Mercedes Espinosa-Lara¹ , Alfonso Méndez-Tenorio¹ and Kenneth L Beattie³

¹  Escuela Nacional de Ciencias Biológicas IPN, México, 11340, D. F., México, 11340, D. F., México

²  Unidad de Investigación Médica en Enfermedades Oncológicas. Hospital de Oncología. Centro Médico Nacional S XXI. IMSS, México, D. F., México

³  Amerigenics, Inc., 1326 Open Range Rd., Crossville, TN 38555 USA

author email corresponding author email

BMC Biotechnology 2005, 5:8doi:10.1186/1472-6750-5-8

Published:	15 February 2005

Abstract

Background

We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes.

Results

18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array.

Conclusion

Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.