OligoArrayDb: pangenomic oligonucleotide microarray probe sets database

OligoArrayDb is a comprehensive database containing pangenomic oligonucleotide microarray probe sets designed for most of the sequenced genomes that are not covered by commercial catalog arrays. The availability of probe sequences, associated with custom microarray fabrication services offered by many companies and cores presents the unequalled possibility to perform microarray experiments on most of the sequenced organisms. OligoArrayDb contains more than 2.8 probes per gene in average for more than 600 organisms, mostly archaea and bacteria strains available from public database. On average, 98% of the annotated genes have at least one probe which is predicted to be specific to its intended target in >94% of the cases. OligoArrayDb is weekly updated as new sequenced genomes become available. Probe sequences, in addition to a comprehensive set of annotations can be downloaded from this database. OligoArrayDb is publicly accessible online at http://berry.engin.umich.edu/oligoarraydb

Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

<div><p>Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining <i>in vitro</i> transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.</p></div

<i>In vitro</i> transcription and reverse transcription (IVT-RT).

<p>(A) Experimental design. (B) 2% agarose gel electrophoresis. An RNA ladder is used as single-stranded ladder. lane A – RNA (step 3), lane B – cDNA (step 5), and lane C – spin-column purified cDNA (step 5).</p

Exonucleolytic hydrolysis of 5<b>′</b>-phosphorylated strand.

<p>(A) Experimental design. (B) lane A – emulsion PCR product (step 2), lane B – exonuclease I hydrolysis of PCR product, lane C – ssDNA product of the lambda exonuclease treatment after removal of PBS (fig. 4 step 6; c), and lane D – exonuclease I hydrolysis of PBS-free ssDNA.</p

Alkaline denaturation method.

<p>(A) Experimental design. (B) lane A – emulsion PCR product (step2), lane B – Nb.BtsI nicked ssDNA (step 3), lane C – nicked DNA (step 4), lane D alkali-melting of nicked ssDNA (step 6), lane E – one of negative selection (step 7), lane F – exonuclease I hydrolysis step 7 products.</p

Primer sequences.

<p>*The restriction enzyme sequence is underlined.</p><p>**T7 promoter sequence is in bold.</p

Removal of universal PCR primer sequences.

<p>(A) Experimental design. (B,C) 2% agarose gel electrophoresis. An RNA ladder is used as single-stranded ladder. lane A – RNA, lane B – sodium hydroxide degradation of RNA, lane C – cDNA (step 1), lane D – exonuclease I hydrolysis of cDNA, lane E – 50 bp ssDNA (step 5; b), and lane E – 50 bp ssDNA (step 6; c), and lane F – exonuclease I hydrolysis of ssDNA.</p

Determination of library coverage.

<p>(a) Experimental design. (b) Distribution of cDNA signal intensity. (c) Venn diagram – Present call per array.</p