DNA ARRAYS

ERDC TN-DOER-C19

December 2000

In order for DNA arrays to be useful as toxicological screening tools, one must know what the vari-

ous mRNAs are signaling the cell to synthesize. Until recently, this information simply did not exist.

However, the human genome project and other genome projects have made great strides in sequenc-

ing and identifying the genetic material in organisms, making it possible to identify huge numbers of

genes and the products they encode. Although the number is rapidly changing due to the intensive

human genome efforts, as of July 2000 over 35,000 human genes have been sequenced and con-

nected to a known gene product, and over 1.7 million Expressed Sequence Tags (ESTs), represent-

ing either novel genes or genes that encode for products homologous but not identical to those of

known genes, have been identified (UniGene human release statistics, July 2000).

There are various approaches to measuring total expressed mRNA, including microarrays and

cDNA arrays. Microarrays, also known as DNA chips, can analyze extremely high numbers of se-

quences. However, these arrays typically include many nucleotide sequences associated with genes

of unknown functions. Since the objective of this work unit (DOER 12B23) was to develop a rapid

and inexpensive screening method for sediment toxicity, it was decided to concentrate on the cDNA

arrays, which use only genes of known function. A cDNA array is composed of a substrate (nylon

membrane, silica chip, or glass slide) upon which nucleotide sequences of known genes are bound to

specific locations (Figure 5). Arrays can be created to analyze anywhere from a few genes to over

50,000 (GeneAlbum, made by Incyte Pharmaceuticals). Briefly, to analyze the relative quantities of

the various genes being expressed, mRNA is extracted from samples (control and treated), con-

verted into cDNA, and then bound to the arrays (Figure 6). Control and treated samples can then be

compared, and the differences in gene expression induced by the treatment can be identified and

quantified.

The cDNA Array Assay. Clontech produces a commercially available cDNA array with up to

1,176 known human genes, grouped by their known functions. Their human toxicology array in-

cludes 588 genes known to be involved with toxicological responses, for example, genes linked to

DNA synthesis/repair, stress responses, and tumor suppression or induction (Figure 5). In order to

monitor responses to toxicant exposure with cDNA arrays, the mRNA is extracted from the samples

and converted into cDNA (Figure 6) using PCR. The extracted mRNA is placed in a tube along with

"primers," or short DNA sequences (oligonucleotides), which bind to complementary sequences on

the mRNA targets. An enzyme then binds to the primer/mRNA complex and makes a complemen-

tary copy of the mRNA sequence. This step increases the stability of the sample, as cDNA is much

more stable than mRNA, which is easily degraded by ubiquitous enzymes (RNases) that are difficult

to eliminate from labware. Additionally, the cDNA can be labeled during this step (radiolabel, fluo-

rescent, or chemiluminescent tags), allowing the detection of the sample after it is bound to the array.

Once the mRNA has been extracted and converted to cDNA, it is ready to be bound (hybridized) to

the cDNA array. The sample is exposed to the array for several hours, during which the cDNA of the

sample binds to that of the array in a complementary sequence-specific manner. The amounts of the

original mRNAs are then quantified by the amount of the label detected at each spot on the array.

Currently, radiolabel (33P) is the most sensitive and most utilized tagging option for mem-

brane-based arrays, although progress is being made for nonradiolabel methods such as fluores-

cence and chemiluminescence. In the Clontech array system, alterations in the mRNA expression

profile are tracked by comparison of two nylon membrane arrays (one control sample, one treated

sample).