Unit –IV
(Omics Techniques)
Gene Expression Analysis: Real-time PCR; Microarray
Differential Protein Analysis: 2D- Gel Electrophoresis
Species Identification: Metagenome Analysis; Barcoding
Chromosome Variability: FISH
Genetic Variability: RAPD; RFLP; AFLP
Gene Editing Tools: CRISPR; TALENS; Zinc Finger Nucleases
Microarray
Microarrays have been developed as a method for rapidly
analyzing the expression of all genes within a genome
(Shalon, Smith, and Brown, 1996).
Microarray in different Context
•Different tissues, same organism (brain v. liver)
•Same tissue, same organism (tumor v. non-tumor)
•Same tissue, different organisms (wt v. mutant)
•Time course experiments (development)
Microarray Methodology
•CHIP Preparation
DNA fragments are physically (Robotic printing)
attached to an inert support (called a chip).
Several different technologies used in Chip
Preparation- Where do the major difference lies?
•the DNA sequences are attached to the chip
•the length of the DNA sequence itself.
Affymetrix –
Photolithograp
hic Technique
Deoxynucleoside
Phosphoramidite
The Affymetrix chips contain chemically synthesized single-stranded DNA
oligonucleotides (∼25 bases in length) that are attached to a quartz surface using
photolabile agents
Affymetrix –
Photolabile
Agents
500 000 individual DNA molecules being placed precisely within a 1.28 cm2 chip
constructed by robotic spotting of DNA fragments, derived from the PCR
amplification of entire genes, onto precise points of a glass slide.
(The yeast genome 6116 separate PCR reactions are performed to amplify every
individual gene. These PCR products are then placed onto a glass slide measuring
approximately 2 cm2. Each PCR product is spotted in a precise location.
Consequently, the sequence of the DNA at any individual spot is known.
Once spotted onto the slide, the DNA is dried by heating to 100 ◦C for 2 s, fixed by UV cross-linking, and then
denatured (95 ◦C, 2 min) to make the DNA single-stranded.
The prepared chips are then used as templates for the binding of labelled cDNA fragments.
if the cells grown under condition 1 express a
gene that is not expressed under condition 2,
then the mixed cDNA will only contain the Cy3
labelled version of the corresponding cDNA and
the complementary spot on the microarray will
be labelled green.
Similarly, a gene only expressed in growth
condition 2 will yield a red spot on the array.
Genes expressed at equal levels in both
samples will give rise to both green and red
labelled cDNAs that can both bind to the
complementary sequence on the array. In
this case a yellow spot (mixture of green and
red) will be generated.
DNA microarrays offer the opportunity to analyse the
expression of many thousands of genes in a single
Disadvantages
experiment. They do, however, suffer from a number of
deficiencies.
For example, like all the experiments we have discussed
that
rely
on
nucleic
acid
hybridization,
the
cross-hybridization of highly similar sequences can prove
problematic. The data obtained from the microarray
experiment must therefore be confirmed by
traditional gene expression analysis experiments.
more
Additionally, the overall sensitivity of the microarray experiment is not high.
Changes in the expression of a gene can only usually be detected if changes
by a factor of two or more occur. Subtle changes in expression, which may
have vital physiological roles, may go undetected during microarray analysis.
Of course, the production of some proteins is not regulated at the level of
transcription. For example, the gene encoding the yeast transcriptional
activator Gcn4p is transcribed constitutively, but the production of protein is
controlled at the level of translation in response to amino acid starvation
(Hinnebusch, 1997).
Finally, the sheer amount of data generated by microarrays has led to difficulties in how this
information is analyzed, and stored in an accessible format.
The efforts of many bioinformaticians are currently aimed at presenting such data in a
format that is usable by biologists.
Databases of publicly available raw and normalized microarray data (e.g.
http://www.dnachip.org) allow the inspection of microarrays to see how your favorite gene is
regulated under particular conditions.