Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.
Identifieur interne : 003069 ( Main/Exploration ); précédent : 003068; suivant : 003070Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.
Auteurs : Jaroslaw Letowski [Canada] ; Roland Brousseau ; Luke MassonSource :
- Journal of microbiological methods [ 0167-7012 ] ; 2004.
Descripteurs français
- KwdFr :
- ADN bactérien (analyse), ADN bactérien (isolement et purification), Bacillus thuringiensis (génétique), Composition en bases nucléiques, Endotoxines (génétique), Fluorescence, Hybridation d'acides nucléiques, Hémolysines, Masse moléculaire, Mésappariement de bases, Protéines bactériennes (génétique), Réaction de polymérisation en chaîne, Sensibilité et spécificité, Sondes d'ADN (), Séquençage par oligonucléotides en batterie (), Température de transition, Toxines bactériennes.
- MESH :
- analyse : ADN bactérien.
- génétique : Bacillus thuringiensis, Endotoxines, Protéines bactériennes.
- isolement et purification : ADN bactérien.
- Composition en bases nucléiques, Fluorescence, Hybridation d'acides nucléiques, Hémolysines, Masse moléculaire, Mésappariement de bases, Réaction de polymérisation en chaîne, Sensibilité et spécificité, Sondes d'ADN, Séquençage par oligonucléotides en batterie, Température de transition, Toxines bactériennes.
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
Affiliations:
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Le document en format XML
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<term>Base Composition</term>
<term>Base Pair Mismatch</term>
<term>DNA Probes (chemistry)</term>
<term>DNA, Bacterial (analysis)</term>
<term>DNA, Bacterial (isolation & purification)</term>
<term>Endotoxins (genetics)</term>
<term>Fluorescence</term>
<term>Hemolysin Proteins</term>
<term>Molecular Weight</term>
<term>Nucleic Acid Hybridization</term>
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<term>Polymerase Chain Reaction</term>
<term>Sensitivity and Specificity</term>
<term>Transition Temperature</term>
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<term>ADN bactérien (isolement et purification)</term>
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<term>Composition en bases nucléiques</term>
<term>Endotoxines (génétique)</term>
<term>Fluorescence</term>
<term>Hybridation d'acides nucléiques</term>
<term>Hémolysines</term>
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<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>
<term>Température de transition</term>
<term>Toxines bactériennes</term>
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<term>Hemolysin Proteins</term>
<term>Molecular Weight</term>
<term>Nucleic Acid Hybridization</term>
<term>Polymerase Chain Reaction</term>
<term>Sensitivity and Specificity</term>
<term>Transition Temperature</term>
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<term>Masse moléculaire</term>
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<term>Réaction de polymérisation en chaîne</term>
<term>Sensibilité et spécificité</term>
<term>Sondes d'ADN</term>
<term>Séquençage par oligonucléotides en batterie</term>
<term>Température de transition</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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