MersV1, Ncbi, Merge, bibRecord, 000274

Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.

Identifieur interne : 000274 ( Ncbi/Merge ); précédent : 000273; suivant : 000275

Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.

Auteurs : Jaroslaw Letowski [Canada] ; Roland Brousseau ; Luke Masson

Source :

Journal of microbiological methods [ 0167-7012 ] ; 2004.

RBID : pubmed:15063067

Descripteurs français

English descriptors

KwdEn :
- Bacillus thuringiensis (genetics), Bacterial Proteins (genetics), Bacterial Toxins, Base Composition, Base Pair Mismatch, DNA Probes (chemistry), DNA, Bacterial (analysis), DNA, Bacterial (isolation & purification), Endotoxins (genetics), Fluorescence, Hemolysin Proteins, Molecular Weight, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis (methods), Polymerase Chain Reaction, Sensitivity and Specificity, Transition Temperature.
MESH :
- chemical , analysis : DNA, Bacterial.
- chemical , chemistry : DNA Probes.
- chemical , genetics : Bacterial Proteins, Endotoxins.
- genetics : Bacillus thuringiensis.
- chemical , isolation & purification : DNA, Bacterial.
- methods : Oligonucleotide Array Sequence Analysis.
- chemical : Bacterial Toxins, Base Composition, Base Pair Mismatch, Fluorescence, Hemolysin Proteins, Molecular Weight, Nucleic Acid Hybridization, Polymerase Chain Reaction, Sensitivity and Specificity, Transition Temperature.

Abstract

DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.

DOI: 10.1016/j.mimet.2004.02.002
PubMed: 15063067

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Le document en format XML

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<term>Sensitivity and Specificity</term>
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<term>ADN bactérien (isolement et purification)</term>
<term>Bacillus thuringiensis (génétique)</term>
<term>Composition en bases nucléiques</term>
<term>Endotoxines (génétique)</term>
<term>Fluorescence</term>
<term>Hybridation d'acides nucléiques</term>
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<term>Masse moléculaire</term>
<term>Mésappariement de bases</term>
<term>Protéines bactériennes (génétique)</term>
<term>Réaction de polymérisation en chaîne</term>
<term>Sensibilité et spécificité</term>
<term>Sondes d'ADN ()</term>
<term>Séquençage par oligonucléotides en batterie ()</term>
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<term>Nucleic Acid Hybridization</term>
<term>Polymerase Chain Reaction</term>
<term>Sensitivity and Specificity</term>
<term>Transition Temperature</term>
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<term>Hybridation d'acides nucléiques</term>
<term>Hémolysines</term>
<term>Masse moléculaire</term>
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<term>Sensibilité et spécificité</term>
<term>Sondes d'ADN</term>
<term>Séquençage par oligonucléotides en batterie</term>
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<term>Toxines bactériennes</term>
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<front><div type="abstract" xml:lang="en">DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.</div>
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<Abstract><AbstractText>DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.</AbstractText>
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Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.

Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.

Source :

Descripteurs français

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Links to Exploration step

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri

Pour générer des pages wiki