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Clinical Chemistry 46: 1555-1561, 2000;
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(Clinical Chemistry. 2000;46:1555-1561.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Evaluation of the Performance of a p53 Sequencing Microarray Chip Using 140 Previously Sequenced Bladder Tumor Samples

Friedrik P. Wikman1, Ming-Lan Lu2, Thomas Thykjaer1, Sanne H. Olesen1, Lars D. Andersen1, Carlos Cordon-Cardo2 and Torben F. Ørntoft1,a

1 Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, 8200 Aarhus N, Denmark.

2 Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
a Address correspondence to this author at: Department of Clinical Biochemistry, Skejby Sygehus, Brendstrupgaardsvej, 8200 Aarhus N, Denmark. Fax 45-89-49-60-18; e-mail orntoft{at}kba.sks.au.dk

Background: Testing for mutations of the TP53 gene in tumors is a valuable predictor for disease outcome in certain cancers, but the time and cost of conventional sequencing limit its use. The present study compares traditional sequencing with the much faster microarray sequencing on a commercially available chip and describes a method to increase the specificity of the chip.

Methods: DNA from 140 human bladder tumors was extracted and subjected to a multiplex-PCR before loading onto the p53 GeneChip from Affymetrix. The same samples were previously sequenced by manual dideoxy sequencing. In addition, two cell lines with two different homozygous mutations at the TP53 gene locus were analyzed.

Results: Of 1464 gene chip positions, each of which corresponded to an analyzed nucleotide in the sequence, 251 had background signals that were not attributable to mutations, causing the specificity of mutation calling without mathematical correction to be low. This problem was solved by regarding each chip position as a separate entity with its own noise and threshold characteristics. The use of background plus 2 SD as the cutoff improved the specificity from 0.34 to 0.86 at the cost of a reduced sensitivity, from 0.92 to 0.84, leading to a much better concordance (92%) with results obtained by traditional sequencing. The chip method detected as little as 1% mutated DNA.

Conclusions: Microarray-based sequencing is a novel option to assess TP53 mutations, representing a fast and inexpensive method compared with conventional sequencing.




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