DNA microarray

1. Presented by
Tejaswini Uppuluri (PALB9109)
Department of seed science and technology

4. A DNA microarray (also commonly known as DNA Chip or biochip) is a
collection of microscopic DNA spots attached to a solid surface.
Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence,
known as probes (or oligos).
Each known gene or “probe” occupies a particular “spot” on the chip, and varying
levels of fluorescent activity show varying levels of gene activity in introduced genetic
material.
Fluorescently labeled target sequences that bind to a probe sequence generate a
signal.

5. Historical background : Evolved from southern blotting
The concept of DNA microarrays began in the mid 1980s.
• Pin based robotic system was developed by Lehrach’s group in 1990.
•“Quantitative Monitoring of Gene Expression Patterns with a complementary
DNA microarray” reported by Patrick Brown, Mark Schena and colleagues in
Science (1995).
• Steve Fodor developed scanner for reading the output.
• Mark schena was proclaimed as the “Father of Microarray
Technology”.
Steve Fodor
Mark schenaPatrick Brown
•
•
•

6. • 1991 - Photolithographic printing (Affymetrix)
• 1994 - First cDNA collections are developed at Stanford.
• 1996 - Commercialization of arrays (Affymetrix)
• 1997- Genome-wide expression monitoring in S. cerevisiae (yeast)
• 2003 - Introduction to clinical practice
• 2004-Whole human genome on one microarray
Singh, A., & Kumar, N. (2013). A review on DNA microarray Technology. International
Journal of Current Research and Review, 5(22), 01-05.

7. The core principle behind microarrays is
hybridization.
Samples are labelled using fluorescent dyes.
At least two samples are hybridized to chip.
Complementary nucleic acid sequences
get pair via hydrogen bonds.
Washing off of non-specific bonding
sequences .
Sample
Preparation and
labelling
Hybridisation Washing
Image acquisition
and data analysis

8. There are 2 types of DNA Chips/Microarrays:
1. cDNA based microarray (or) Spotted DNA arrays
2. Oligonucleotide based microarray

9. There are certain requirements for designing a
DNA microarray system viz.,:
1. DNA Chip
2. Target sample (Fluorescently labelled)
3. Fluorescent dyes
4. Probes
5. Scanner

10. Various types of cells in body are different from one another due to different genes being
“turned on” and “turned off”. Because of differ in GENE EXPRESSION
2. Tissue samples dissolved in organic solvent .RNA extracted.
DNA, proteins and other cell components separated.
1. Sample extraction from healthy and unhealthy tissue of same
plant.
Healthy Unhealthy
Mixing the tissue samples on the Vortex. RNA will be
released. Samples are centrifuged to separate RNA
from rest of the cell components

11. • After centrifugation, RNA separated from the rest.
Then both samples are pipetted out in different
collection tubes
• RNA Samples washed over Columns filled
with small beads
• These beads bind only to RNA strands with
Poly-A tail
• Other molecules washed away
Healthy Unhealthy
Healthy Unhealthy
Healthy Unhealthy
• Wash Buffer solution over beads
• mRNA detached from the beads
Healthy Unhealthy
• DNA copy of the RNA is made giving it
some colour as labelling mix
• Green--Healthy cells RNA
• Red - infected cells RNA

12. • Reverse transcriptase assembles
labelled nucleotides into a cDNA
molecule
• mRNA molecule degraded.
Both the Samples are spread on the Microarray

13. • Microarray placed in washing solution. Extra cDNA
present on slide are washed off easily as it won’t bind.
HEALTHY CELLS
• Many of the spots are Green but not every
• Dark Spots are genes not transcribed in Healthy cells
CANCER CELLS
• Red Spots pattern looks different
• Infected cells and Healthy skin cells have different
gene expression pattern
RED AND GREEN SPOTS MERGED TOGETHER
• Yellow spot –gene expressed both in healthy cells and unhealthy cells
• Yellow genes—No change in activity when cell becomes unhealthy
• Red spots--genes “turnedon” in unhealthy cells
producing more mRNA
• Green spots –genes “turned-off’ in unhealthy cells

14. Microarrays use relative quantization in which intensity if spot is compared to the same spot
Under a different condition which is known by its position
• The two samples to be compared (pair wise
comparison) are grown/acquired.
• RNA, DNA, DNA/RNA bound to a protein
• Optional PCR Amplification
• The label is added either in the RT step or in an
additional step after amplification if present.
• This mix is denatured and added to a pin hole in a
microarray.
• The holes are sealed and the microarray hybridized.
• The microarray is dried and scanned in a special
machine where a laser excites the dye and a detector
measures its emission. After that the raw that is
normalized for study

16. Gene Chips
1. Oligonucleotide arrays (Affymetrix)
 Small number of 20-25mers/gene
 Enabled by photolithography from the computer industry
 Off the shelf
2. Ink-jet microarrays (Agilent)
 Large number of 25-60mers “printed” directly on glass
 Four cartridges: A, C, G, and T
 Flexible, rapid, but expensive.

17. Spotted Array Affey gene chips
Relative cheap to make (~$10 slide) Expensive ($500 or more)
Flexible - spot anything you want Limited types available, no chance
of specialized chips
Cheap so can repeat
experiments many times
Fewer repeated experiments
usually
Highly variable spot
deposition
More uniform DNA features
Usually have to make your
own
Can buy off the shelf