Gene representative

Fig. 3. Constmetion of a recombinant DNA by joining vector and passenger fragments where (I I I I I I) represent sticky ends. Both A and B genes represent selectable traits, so that introduction of foreign DNA in B gene leads to the loss of an identifiable function. REP represents repHcation and...

The homeobox genes represent important nodes in the genetic circuitry of development. They are nevertheless but one set of links in the chain of genetic events, and to understand their function it is necessary to determine how they are regulated and how they in turn regulate downstream genes. 

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

The issue of whether to use the enriched poly(A) RNA or total RNA was addressed in a later paper by Mahadevappa and Warrington (1999). Using human adenocarcinoma cells, they examined the recovery of detectable transcripts from varying numbers of cells for both protocols. Of the -1800 genes represented on the chip, about 35% were observed to be detectable using either of the two preparahons. 

The basic dogma of biology states that information flows from genes, represented by DNA, via a messenger molecule, the mRNA, to proteins ... 

Microarrays based on cDNA or oligonucleotides differ fundamentally in how their experimental outputs are analyzed. Typical cDNA arrays are hybridized with differentially labeled cDNA pools generated from two separate RNA sources. For example, RNA may be harvested from untreated cells grown under a standard set of conditions and the cDNA produced from this RNA pool may be labeled with one fluorescent dye. A second RNA sample is then harvested from the same cell type after it is treated with a chemical or grown under a different set of conditions, and the cDNA is labeled with a second dye. Once the two cDNAs can be distinguished by their labels, equal amounts are mixed and hybridized competitively to the same cDNA array. The ratio of one dye signal to the other at each probe will reflect relative differences in abundance between the two RNA samples for the gene represented. 

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