PCR-Based Setup for High-Throughput cDNA Library Sequencing on the ABI 3700™Automated DNA Sequencer

Smith, Timothy P. L.; Godtel, Renée A.; Lee, Robert T.

doi:10.2144/00294bm05

Cited by 23 publications

(18 citation statements)

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“…Amplification was verified by running PCR products on a 1% agarose gel containing ethidium bromide. DNA exonuclease digestions for Sanger sequencing were set up using a modified 2.0 BigDye protocol (Applied Biosystems, Carlsbad, CA) (59). For the initial reaction, 5.5 l of the PCR product and 7 l exonuclease I (ExoI) (0.1 U/l) were added to each well.…”

Section: Strain Collectionmentioning

confidence: 99%

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Norman

Strockbine

Bono

2012

Appl Environ Microbiol

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ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains are important food-borne pathogens capable of causing hemolytic-uremic syndrome. STEC O157:H7 strains cause the majority of severe disease in the United States; however, there is a growing concern for the amount and severity of illness attributable to non-O157 STEC. Recently, the Food Safety and Inspection Service (FSIS) published the intent to regulate the presence of STEC belonging to serogroups O26, O45, O103, O111, O121, and O145 in nonintact beef products. To ensure the effective control of these bacteria, sensitive and specific tests for their detection will be needed. In this study, we identified single nucleotide polymorphisms (SNPs) in the O-antigen gene cluster that could be used to detect STEC strains of the above-described serogroups. Using comparative DNA sequence analysis, we identified 22 potentially informative SNPs among 164 STEC and non-STEC strains of the above-described serogroups and designed matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) assays to test the STEC allele frequencies in an independent panel of bacterial strains. We found at least one SNP that was specific to each serogroup and also differentiated between STEC and non-STEC strains. Differences in the DNA sequence of the O-antigen gene cluster corresponded well with differences in the virulence gene profiles and provided evidence of different lineages for STEC and non-STEC strains. The SNPs discovered in this study can be used to develop tests that will not only accurately identify O26, O45, O103, O111, O121, and O145 strains but also predict whether strains detected in the above-described serogroups contain Shiga toxin-encoding genes.

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Section: Strain Collectionmentioning

confidence: 99%

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Norman

Strockbine

Bono

2012

Appl Environ Microbiol

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“…The PCR was performed in a PTC-200 DNA engine (MJ Research Inc., Watertown, MA) using 0.5 U of HotStarTaq polymerase (Qiagen, Valencia, CA), 1× of supplied buffer with 1.5 mM MgCl 2 , 200 µM dNTP, 0.8 µM each primer, and 100 ng of genomic DNA in 25-µL reactions. Five microliters of the PCR were electrophoresed in 1.5% agarose gels to determine quality of amplification and the remainder was treated with 0.1 U of exonuclease I (USB Corp., Cleveland, OH) for 30 min, precipitated with isopropanol, and resuspended in water for sequencing on an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA; Smith et al, 2000). The PCR products were sequenced directly using the original amplification primers.…”

Section: Snp Marker Development and Genotypingmentioning

confidence: 99%

Genetic relationships of body composition, serum leptin, and age at puberty in gilts12

Kuehn

Nonneman

Klindt

et al. 2009

Journal of Animal Science

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“…Individual colonies were picked into 384-well plates with a robot from plated aliquots of each library by BACPAC resources. Single-pass sequencing was performed as described by Smith et al (2000) using PCR amplification of inserts for template preparation and SP6 sequencing primer. Sequences of sufficient length and quality for submission to GenBank have been deposited in the dbEST database.…”

Section: Est Sequencingmentioning

confidence: 99%

Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle

Smith¹,

Grosse²,

Freking³

et al. 2001

Genome Res.

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100

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An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter-and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.The rapid progress of genomic research in diverse organisms such as yeast, fruit flies, nematodes, mice, and humans has been driven by a combination of mapping, sequencing, and identification of expressed portions of each genome. Progress in these areas has lagged in the livestock species, limiting the use of functional genomics approaches to current problems in production-animal agriculture. Resources for a public effort to sequence the genomes of livestock species are not currently available. However, more modest sequencing efforts aimed at cDNA libraries have substantial value to the research community, especially when combined with mapping efforts to produce comparative maps with other mammalian species. Comparative maps make use of the general conservation of synteny between mammals and allow the livestock community to tap into the wealth of information generated in the human genome effort.To provide a resource of livestock-specific expressed sequence data to facilitate proteomics and functional genomics approaches in animal science, an EST-sequencing program was initiated with the aim to maximize the efficiency of obtaining sequence from the highest possible number of independent genes. Four normalized bovine cDNA libraries specifically designed for this task were produced and arrayed for high-throughput sequencing. To make the data more accessible and useful, assembly and annotation analyses were performed and an interactive Web site constructed by The Institute for Genomic Research (TIGR) to view and analyze bovine genes. We report the construction of this Cattle Gene Index and assessment ...

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PCR-Based Setup for High-Throughput cDNA Library Sequencing on the ABI 3700™Automated DNA Sequencer

Cited by 23 publications

References 0 publications

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Association of Nucleotide Polymorphisms within the O-Antigen Gene Cluster of Escherichia coli O26, O45, O103, O111, O121, and O145 with Serogroups and Genetic Subtypes

Genetic relationships of body composition, serum leptin, and age at puberty in gilts12

Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle

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