Biomedical Engineering Reference
In-Depth Information
Serial analysis of gene expression (SAGE)
SAGE is a method that was designed to provide a relative quantitation
and unbiased analysis of the genes expressed in a cell rather than dif-
ferences between cells. It involves the production of short (14 bp) cDNA
fragments upstream the poly(A) tail of the expressed mRNA, a size that
has been calculated to be sufficient to identify the gene from which
it is expressed. These tags are concatemerized, cloned and amplified
in bacteria. These cloned tags can then be sequenced “en masse” to
identify the expressed genes in the cell.
DNA microarrays
While other methods for accomplishing differential screening de-
scribed above are still useful, the advent of DNA microarrays, also
known as DNA chips or gene chips, have revolutionized the ability to
rapidly identify differences in the expression of genes between differ-
ent cells and tissues. DNA microarrays are arrays containing a large
number of samples of DNA that represent many if not all of the known
genes in a given species, tethered to the array matrix in a known order.
The DNA samples on the matrix are either cDNAs (in sizes of a few
hundred to a few thousand bp) or oligonucleotides 20-80 bp in length
representing each of the known genes. The matrix is usually glass, but
nylon substrates are also used. Current technology using robotic fabri-
cators allows for the covalent attachment of 30,000 to 60,000 individual
oligonucleotides to a single matrix, providing (in theory) one or more rep-
resentatives of each of the thousands of expressed genes in the human
genome. DNA microarrays are available for the human, mouse and rat
genomes, and microarrays representing a number of pathogens have
also been produced.
The microarrays are probed with cDNAs that have been synthesized
from a sample of RNA incorporating a fluorescent nucleotide. The la-
beled cDNA can then be used to probe the matrix, and the hybridized
species are detected by an sophisticated array reader to determine
which of the genes on the matrix are expressed in the RNA sample. Since
the genes of any species can be represented on one or two microarrays,
an investigators can now determine which genes are expressed in any
given cell or tissue within the matter of a couple of days!
However, the capabilities and uses of DNA microarrays are even
greater! With the help of powerful computer programs (the analysis of
microarray data is computationally intensive and generates more data
than can be easily interpreted without the aid of computers), information
from DNA microarrays can be used to compare the expressed genes in
Search WWH ::
Custom Search