New GeneConnection discovery microarrays provide more than 4,000 unique human cDNA sequences
Stratagene introduces the first in a series of human cDNA microarrays on glass slides. These GeneConnection discovery microarraysII which are based on the technology published by Schena et al.,1 can be used for monitoring RNA expression levels of genes. The microarrays are configured on standard 25 mm x 75 mm glass microscope slides. Each slide contains more than 4,000 human cDNA sequences spotted in a 44 x 96 grid.
The human cDNA spotted on the GeneConnection discovery-3 microarrays are from Stratagenes GeneConnection discovery clone collection. This collection consists of clones from our innovative libraries and contains a high number of clones that do not have significant nucleotide homology to clones in public databases.* Therefore using Stratagenes human cDNA microarrays allows gene expression profiling to be combined with gene discovery to maximize results.
All of the cDNAs in the discovery clone collection, including those used to create the human cDNA microarrays, are available. Approximately 300 to 600 bases at the 3 end of each of these cDNAs have been sequenced in a single pass. The sequences were compared to those in the public databases using the Basic Local Alignment Search Tool (BLAST) technology. Clones having significant nucleotide homology to one or more characterized sequences in the public databases are assigned a name, accession number, and UniGene number. Clones having significant homology to one or more Expressed Sequence Tag (EST) sequences in the public databases are also assigned an EST number. Clones not having significant nucleotide homology to those in the public databases are identified as unknown o n the Stratagene website.
Several methods are available to identify and evaluate the clones in the discovery clone collection. The collection can be searched for a specific clone by using a gene name, accession or UniGene number, nucleotide sequence, or location on a discovery-3 microarray. Additional information on the Virtual Lab website includes gel images of restriction enzyme digestions of individual clones and gel images demonstrating the length and purity of PCR products used for microarray spotting (Figure 1). Sequence information is available upon purchase of any clone.
Most cDNA microarrays are created by spotting small amounts of PCR products obtained from plasmid cDNA templates onto glass microscope slides. Such PCR products are typically generated using two vector-specific primers that anneal to priming sites flanking the cDNA insert. There have been many reports that the DNA spotted onto such microarrays is often a mixture of more than one clone or an incorrect clone.
Stratagene has introduced several quality-control steps to essentially eliminate the spotting of more than one clone per spot or spotting an incorrect clone on the discovery-3 microarrays. These steps include digestion of each plasmid cDNA template with restriction enzymes to determine the DNA purity and nucleotide sequencing from the 3 end (Figure 2). The 3 sequence information is then used to design an insert-specific primer that is capable of amplifying approximately 350 bases of the 3 end of the cDNA, including the polyA tail. After PCR with the insert-specific primer, the presence of a single PCR product of the correct length is confirm ed by gel electrophoresis. If the cDNA template contained minor amounts of contaminating DNA, such DNA will not amplify with the insert-specific primer. Moreover, if the cDNA templates have been mixed-up in a prior step, a PCR product of the predicted length will not be amplified. Thus, PCR with an insert-specific primer both purifies and confirms the identity of the cDNA.
Genes are often grouped into families based upon regions of protein homology and, frequently the nucleotide sequences of the protein coding regions have sufficient nucleotide homology to cross hybridize. In addition, 3 untranslated regions sometimes contain Alu sequences and other repeat elements which can cross hybridize. For these reasons, the 3 insert-specific primer was designed to amplify a portion of the cDNA including little or no coding region and excluding repeat elements. Hence, this design increases hybridization specificity when using the discovery-3 microarray by minimizing the chances that the DNA in any given spot will cross hybridize with RNA-derived probes from other gene family members or with repeat elements.
Each GeneConnection discovery-3 microarray has control spots distributed throughout the microarray in a defined pattern to assist in quality control and orientation of the microarray. There are five types of control spots: spotting buffer only, human -actin DNA, and three Arabidopsis thaliana DNA. The A. thaliana genes were selected because they are involved in plant-specific processes and do not have known human homologues. In addition, they do not hybridize to membrane-arrayed human cDNA from 29 different tissues. In gene expression profiling experiments, the human -actin gene should hybridize with most, if not all, human R NA-derived probes. In contrast, the buffer spot and A. thaliana DNA should not hybridize to any human RNA-derived probe. RNA transcripts of these three A. thaliana genes and a Control Microarray are available separately.
The discovery clone collection and discovery-3 microarray offer unique and valuable tools for combining gene expression profiling with gene discovery. The high number of unknown and uncharacterized clones in the clone collection and on the microarrays greatly increases the chances for novel gene identification. The unique design of the discovery-3 microarrays increases the probability of identifying specific genes. Stratagene will release new discovery microarrays at regular intervals and welcomes the use of our website to find clones of interest.
Schena, M., et al. (1995) Science 270: 467-470.
* Patents pending