Page 63 - Clinical Biochemistry 08PB804

• Basic idea involves hybridization (annealing) between the cDNAs (reverse DNAs)
obtained by reverse transcription of mRNAs obtained from the target sample cells to pre-
designed (synthesized) complementary DNA probe arranged on an array.

• A microarray, therefore, consists of a pre-designed library of synthetic DNA probes that
are arrayed on a solid matrix.

DNA Microarray Technique

1. When a gene is activated, certain segments of the gene are copied to form mRNA, which
is the template for protein synthesis in the cell.

2. mRNA is complementary to the original portion of the DNA strand (i.e. Gene sequence)
from which it was copied.

3. A database of over 40,000 gene sequences is available, that can be used on a solid array.

Identification of Active/Inactive Genes

a) mRNA (isolated & purified) from cells of interest, one sample serves as control and the
other as the experiment (i.e. Healthy vs disease).

b) mRNA is treated with the enzyme reverse transcriptase and labeled by a fluorescent dye to
get a fluorescent-cDNA (disease & healthy samples can be labeled with different dyes).

c) Hybridization of the labeled cDNA to the microarray: This step involves placing cDNAs
onto a DNA microarray, where it will hybridize to their synthetic complementary DNA probes
attached on the microarray leaving its fluorescence tag. A series of washes are used to remove
non-bound sequences.

d) Scan the microarray and quantitate the signal: The fluorescent tags of cDNA are excited by
a laser signal. The total strength of the signal depends upon the amount of target sample binding
to the probes present on that spot. The amount of target sequence bound to each probe correlates
to the expression level of various genes expressed in the sample. The signals are detected,
quantified and used to create a digital image of the array.

e) If a particular gene is very active, it produces many molecules of mRNA, thus more labeled
cDNAs, which hybridize to the DNA on the microarray slide and generate a very bright
fluorescent area.