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

Letowski, Jaroslaw; Brousseau, Roland; Masson, Luke

doi:10.1016/j.mimet.2004.02.002

Cited by 116 publications

(105 citation statements)

References 39 publications

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“…To minimize false-positive results, a consentaneous approach was incorporated where multiple gene probes must produce positive hybridizations to confirm the presence of that gene. Since cry genes at increasingly higher ranks possess similar sequences, it becomes increasingly difficult to design specific cry gene probes to differentiate between tertiary-and quaternary-level genes inasmuch as five or more mismatches distributed over a 50-base sequence are required to prevent cross-hybridization (26). For example, since one cannot find a specific probe for cry1Ic, two probes were designed.…”

Section: Discussionmentioning

confidence: 99%

“…Lack of potential hairpin formation within each probe was verified using mFold v.3.0 software (44). Based on oligonucleotide design criteria (26), 101 oligonucleotides were synthesized (IDT, Coralville, IA) for printing on the microarray. The melting point temperature (T m ) of each probe, as calculated according to the formula optimized for DIG Easy Hyb digoxigenin buffer, where T m ϭ 49.82 ϩ 0.41 ϫ (%GϩC) Ϫ 600/probe length in base pairs, was kept between 50 and 58°C.…”

Section: Methodsmentioning

confidence: 99%

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Assessment of cry1 Gene Contents of Bacillus thuringiensis Strains by Use of DNA Microarrays

Letowski

Bravo²,

Brousseau

et al. 2005

Appl Environ Microbiol

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A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primarybut not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.The gram-positive bacterium Bacillus thuringiensis produces one or more insecticidal crystal proteins (Cry) in the form of an intracellular parasporal crystal (37). After ingestion by a susceptible insect, Cry proteins dissolve in the insect midgut, where most are subsequently activated by midgut proteases. The protease-resistant toxin binds to specific docking proteins on the microvillous surface of susceptible midgut epithelial cells and then oligomerizes (35). Finally, the oligomeric toxin inserts into the membrane, forming a pore (9). To date, the structure of the internal Cry pore in the membrane remains uncertain; however, studies with synthetic membranes show that during membrane integration, the toxin undergoes a conformational change in which the helix-rich domain I separates from domains II and III (38) and a hairpin, composed of helices ␣4 and ␣5 subsequently inserts into the membrane with ␣4 lining the pore lumen to create a functional ion channel (34).

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Assessment of cry1 Gene Contents of Bacillus thuringiensis Strains by Use of DNA Microarrays

Letowski

Bravo²,

Brousseau

et al. 2005

Appl Environ Microbiol

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“…Finally, it is interesting that the effect of ⌬G°d uplex on the trade-off between probe sensitivity and specificity is analogous to the effect of probe length. In general, long probes (50-to 70-mers) are more sensitive than short probes (15-to 30-mers) but display lower specificity (28,48). Cost and flexibility in microarray probe synthesis are often recognized as two of the major limiting factors in the use of microarray technology for diagnostic purposes (6,21).…”

Section: Vol 74 2008 In Situ-synthesized Vmg Biochip For Pathogens mentioning

confidence: 99%

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water

Miller¹,

Tourlousse²,

Stedtfeld³

et al. 2008

Appl Environ Microbiol

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Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately ؊19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

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“…These results were achieved by using a 30-mer oligonucleotide probe allowing a more specific detection of the target gene. Previous studies that assessed optimal conditions for fluorescent detection on DNA microarrays demonstrated that the use of short, 30-mer probes were equivalent to background noise, whereas optimal hybridization signals required 70-mer or 100-mer oligonucleotide probes (Letowski et al, 2004). However, the use of longer probes resulted also in lower specificity due to cross-hybridization with nontarget genes in some species (Letowski et al, 2004).…”

mentioning

confidence: 99%

“…Previous studies that assessed optimal conditions for fluorescent detection on DNA microarrays demonstrated that the use of short, 30-mer probes were equivalent to background noise, whereas optimal hybridization signals required 70-mer or 100-mer oligonucleotide probes (Letowski et al, 2004). However, the use of longer probes resulted also in lower specificity due to cross-hybridization with nontarget genes in some species (Letowski et al, 2004). In the present study, the use of 30-mer probes on the microarray in combination with photopolymerization facilitated the detection of specific target regions in the genes selected for identifying E. coli O157.…”

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confidence: 99%

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

Quiñones

Swimley

Taylor

et al. 2011

Foodborne Pathogens and Disease

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Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 AE 7.12 to 76.71 AE 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU=mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

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

Cited by 116 publications

References 39 publications

Assessment of cry1 Gene Contents of Bacillus thuringiensis Strains by Use of DNA Microarrays

Assessment of cry1 Gene Contents of Bacillus thuringiensis Strains by Use of DNA Microarrays

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

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