Biomedical Engineering Reference
In-Depth Information
ics, it is generally assumed that the genes discussed have been previously se-
quenced and placed in a computer database.
2.
Gene expression analysis
.
Expression analysis
attempts to determine
which genes are being expressed in a given tissue or cell type at a specific mo-
ment in time. The objective, to identify all the genes that are being expressed, is
challenging because of the great complexity of the mixture of mRNA being ana-
lyzed—each cell may express as many as tens of thousands of genes. SAGE
Tagging and cDNA hybridization arrays, as discussed below, are techniques for
determining comprehensive gene expression data for a given cell type or tissue.
The technique of differential expression analysis compares the level of gene
expression between two different samples. Variations in the level of expression
of individual genes or groups of genes can provide valuable clues to the underly-
ing mechanism of the disease process. There are a number of methods currently
used to obtain comprehensive gene expression data.
a.
cDNA hybridization arrays
. A cDNA hybridization array is composed of
distinct DNA strands arrayed at spatially distinct locations. A cDNA hybridiza-
tion array operates by hybridizing the array with fluorescent-tagged probes made
from mRNA, which anneal to its DNA strands. This generates a fluorescent im-
age-defining expression, which provides a very rapid optical readout of ex-
pressed genes. However, cDNA hybridization arrays are generally manufactured
for use with a given set of expressed genes, for example, those of a given
cell type. The design and manufacture of cDNA hybridization arrays for a
given expression library of a size over 10,000 can be quite costly and lengthy.
Affymetrix has recently developed an oligonucleotide array, known as a Univer-
salChip, that is not specialized to any gene library; it consists of 2000 unique
probe sequences that exhibit low cross-hybridization and broad sampling of se-
quence space It can be used with fluorescent-tagged probes made from DNA
rather than mRNA. This technology can be used for output in a Biomolecular
Database system.
b.
Serial analysis of gene expression
(SAGE) is a technique for profiling the
genes present in a population of mRNA. By the use of various restriction en-
zymes, SAGE generates, for each mRNA, a 10-base tag that usually uniquely
identifies a given gene. In the usual SAGE protocol, the resulting SAGE tags are
blunt-end ligated together and the results are sequenced. The sequencing is
faster than sequencing the entire set of expressed genes because the tags are
much shorter than the actual mRNA they represent. Once sequencing is com-
plete, the tag sequences can be looked up in a public database to find the corre-
sponding gene. Using the sequence data and the current UniGene clusters, a
computer processing stage determines the genes that have been expressed.
SAGE can be used on any set of expressed genes and it is not specialized to a
particular set. This technology can be adapted for use for additional information
tags appended to the DNA in a Biomolecular Database. Genzyme Molecular
Oncology Inc. is the developer of this SAGE technology.
