Reliable Hybridization of Oligonucleotides as Short as Six Nucleotides

Drmanac, Radoje; Strezoska, Žaklina; Labat, Ivan; Drmanac, Snezana; Crkvenjakov, Radomir

doi:10.1089/dna.1990.9.527

Cited by 63 publications

(35 citation statements)

References 15 publications

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“…Hybridization solution (1.8 ml of 5% sarcosyl-0.2 M Na phosphate [pH 7.2] containing 1 nM DNA oligonucleotide probe end labeled with 33 P) was applied to the membranes, which were covered with plastic sheeting (thickness, 102 m), and incubated overnight at 12°C (11). The arrays were washed twice in 0.1ϫ to 4ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 to 30 min at 12°C (12); the wash conditions were determined empirically for each probe.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

Valinsky¹,

Vedova

Scupham³

et al. 2002

Appl Environ Microbiol

120

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One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa: Bacillus, Actinobacteria, Proteobacteria, and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.

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Section: Methodsmentioning

confidence: 99%

Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

Valinsky¹,

Vedova

Scupham³

et al. 2002

Appl Environ Microbiol

120

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“…A list of 2,000 nonamer microarray capture probes was generated by random computer selection based on the sequence of the E. coli K-12 genome (GenBank accession number U00096). Because the capture probes are only 9 nucleotides in length, any one probe is expected to occur (on average) once every 131,000 bases in any double-stranded genome (once every 4 9 bp ϭ 262,000 bp; 131,000 bases in a double-stranded sequence). The computer program was written to perform the following screens: any repeated sequence was less than 4 nucleotides; there were no terminal, 3-nucleotide inverted repeats (hairpins); any probe containing a GGGCCC repeat was discarded; the GϩC content was maintained between 44 and 55%; and any probe containing a palindrome was eliminated.…”

Section: Methodsmentioning

confidence: 99%

Fingerprinting Closely Related Xanthomonas Pathovars with Random Nonamer Oligonucleotide Microarrays

Kingsley

Straub²,

Call

et al. 2002

Appl Environ Microbiol

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Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.The need to rapidly detect specific microorganisms is both varied and extensive, encompassing basic biochemical, genetic, and ecological research and numerous applications in the genetic identification and tracking of pathogenic microorganisms. Current epidemiological investigations of pathogenic microorganisms use fairly standard techniques for DNA fingerprinting or discriminating between closely related isolates. These include pulsed-field gel electrophoresis (2), variations on Southern hybridization (43), and PCR-based techniques such as randomly amplified polymorphic DNA PCR (39), repetitive element PCR (18, 24), analysis of restriction fragment length polymorphisms (20, 30), single-stranded conformation polymorphisms (26), denaturing gradient gel electrophoresis (29), and combinations thereof (40). In most cases, current DNA-typing methods access a limited complement of genetic information and the fingerprint is based on DNA fragment sizing technology (i.e., gels) that requires parallel processing with many independent restriction enzymes or probes to achieve statistical rigor and confidence in the resulting pattern of DNA fragments.Despite the widespread acceptance of gel-based DNA fingerprinting techniques, they frequently fail to answer fundamental epidemiological questions. For example, Hancock et al. identified multiple sources of Escherichia coli O157:H7 in feedlots and dairy farms but were un...

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“…It was developed by a team of Serbian scientists in the early 1990's (35,36). The idea was to determine the oligonucleotide content of the template DNA chain by hybridizing it with short (5-25 bp) oligonucleotides (37).…”

Section: Sequencing By Hybridization (Sbh)mentioning

confidence: 99%

The History of DNA Sequencing / ISTORIJAT SEKVENCIRANJA DNK

Gužvić¹

2013

Journal of Medical Biochemistry

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SummaryDuring the last decade, the cost of DNA sequencing technologies has decreased several orders of magnitude, with the proportional increase of speed and throughput. Human Genome Project took almost 15 years to complete the sequence of the human genome. With the second and third generation technologies, this can be done in the matter of days or hours. This progress and availability of sequencing instruments to virtually every researcher leads to replacing of many techniques with DNA sequencing and opens new venues of research. DNA sequencing is used to investigate basic biological phenomena, and is probably going to be increasingly used in the context of health care (preimplantation diagnostics, oncology, infectious diseases). Current trends are aiming towards the price of 1000$ for sequencing of one human genome. Without any doubt, we can expect improvement of existing and the development of fourth generation technologies in the coming years.Keywords: DNA sequencing; genome; molecular biology Kratak sadr`ajTokom poslednjih 10 godina, cena sekvenciranja DNK se drasti~no smanjila, dok se, suprotno tome, brzina sekvenciranja i veli~ina genoma koja se mo`e sekvencirati u jedinici vremena, pove}ala. Sekvenciranje kompletnog humanog genoma u okviru projekta »Humani Genom« trajalo je skoro 15 godina. Sa dana{njim stepenom razvoja, tehnologijama sekvenciranja druge i tre}e generacije, isti projekat bio bi realizovan za nekoliko dana, pa ~ak i sati. Napredak u tehnologiji sekvenciranja i dostupnost novih tipova instrumenata za sekvenciranje gotovo svakoj laboratoriji rezultirala je mnogo masovnijom upotrebom ove metodologije u analizi DNK kao i otvaranjem novih pravaca u istra`ivanjima. Sekvenciranje DNK se koristi za istra`ivanje osnovnih biolo{kih fenomena, i verovatno }e sve vi{e nalaziti upotrebu u za{titi zdravlja (preimplantaciona dijagnostika, onkologija, infektivne bo lesti). Trenutno, osnovni trend u ovoj oblasti je obaranje cene sekvenciranja po pojedina~nom humanom genomu na 1000$. Nema nikakve sumnje da }e se u narednim godinama pojaviti tehnologije ~etvrte generacije.

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Reliable Hybridization of Oligonucleotides as Short as Six Nucleotides

Cited by 63 publications

References 15 publications

Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

Fingerprinting Closely Related Xanthomonas Pathovars with Random Nonamer Oligonucleotide Microarrays

The History of DNA Sequencing / ISTORIJAT SEKVENCIRANJA DNK

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