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Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides

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Abstract

In this article we introduce a strategy of preanncaling labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous δF508 and heterozygous δF508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon l0 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, δI507, δF508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant allcles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with32P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base slacking interactions between the “capture probe” and the auxiliary oligonucleotide. The presence of the δF508 mutation was delected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mcr probes was improved by increasing the temperature of hybridization from 15 to 25‡C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis:

  1. 1

    A convenient means of introducing label into the target DNA molecule.

  2. 2

    Disruption of interfering short-range secondary structure in the region of analysis.

  3. 3

    Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target).

  4. 4

    Tandem hybridization with the capture probe (providing contiguous stacking) as a means for achieving efficient mismatch discrimination at the terminal position of the capture probe (adjacent to the auxiliary oligonucleotide).

By use of multiple auxiliary oligonucleolides. all of the above benefits can be derived simultaneously.

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Author notes

  1. Kenneth L. Beattie

    Present address: Oak Ridge National Laboratory, 37831-6123, Oak Ridge, TN

Authors and Affiliations

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

    Rogelio Maldonado-Rodriguez, Mercedes Espinosa-Lara & Armando Calixto-Suárez

  2. DNA Technology Laboratory, Houston Advanced Research Center, 77381, The Woodlands, TX

    Wanda G. Beattie & Kenneth L. Beattie

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  1. Rogelio Maldonado-Rodriguez
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Maldonado-Rodriguez, R., Espinosa-Lara, M., Calixto-Suárez, A. et al. Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides. Mol Biotechnol 11, 1–12 (1999). https://doi.org/10.1007/BF02789172

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