Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
Microarray By Pushpita Saha
1. Microarray
ST. THOMAS COLLEGE, BHILAI
SESSION:
2020-21
Paper 9 : Genetic Engineering
Guided by :
Dr. Shubha Diwan
Presented by :
Pushpita Saha
(M.Sc. 3rd Sem Biotechnology)
3. Introduction
• Microarray is a solid surface (membrane or glass slide) to which
biological molecules such as DNA, proteins, etc. are arranged in a
regular pattern in a very concise space.
• It’s a technology that enables to investigate on a large no. of genes
that are very cumbersome using traditional methods.
• Also called as BIO-CHIP or GENE-CHIP.
• Primarily used for
1. Identification of any particular gene sequence.
2. Expression Analysis of genes.
Fig 1: Microarray
Image source:
https://upload.wikimedia.org/wikipedia/commons/0/0e
/Microarray2.gif
4. History
In 1995, “ Quantitative monitoring of gene expression patterns with
complementary DNA microarray” was reported by Mark Schena and his
colleagues .
A complete eukaryotic genome of Saccharomyces cerevisiae on
microarray was published by Pat Brown and his group in 1997.
Mark Schena – The father of Microarray technique
Fig 2: Mark Schena
Image source: https://alchetron.com/cdn/mark-schena-7d54ccd7-324a-494f-b033-ba21fc03c79-resize-750.jpeg
5. Principle
• Based on hybridization principle.
• Each spot contains picomoles of short oligonucleotide sequences spotted
on the array surface called as probes.
• The sample is usually fluorescently labelled by dyes called as
fluorochrome
• Radio-labelling can also be done to label the target sample.
• The sample binds to the probe present on the array spots and
fluorescence caused at the sites of binding generates a signal i.e.
detected.
8. 1. Spotted Microarrays
• First technique in which microarrays were manufactured.
• Arrays are made using spotting robot .
• Pre synthesized cDNA are used as probes.
• Steps of array synthesis are
I. Making of DNA probes
II. Spotting arrays into glass surface using spotting robot.
III. Post-spotting processes.
Fig 3 : Spotting
Image source: Muller U R & Nicolau D V (2005)
11. 2. In- situ Oligonucleotide Arrays
• Instead of pre-synthesized cDNA, here oligos are built up base by base on the
array surface.
• Covalent reaction between ‘OH’ of sugar of last nucleotide to be attached to the
phosphate group of the next nucleotide.
• Each new nucleotide added consists of a protective group (PG) on 5’ position.
• This PG is converted into OH group by acid or light.
Fig 6(b) : Oligonucleotide array synthesis
Image source – Stekel , D. (2003)
12. Technologies for making in-situ Oligonucleotide Synthesis
1. Affymetrix
Technology
3. Inkjet
Array
Technology
2. Mask-less
photo-
deprotection
Technology
17. Figure 12 : Summary of Microarray Process
https://microbenotes.com/wp-content/uploads/2018/09/DNA-Microarray.jpg
Image source -
18. 1. ARRAY PRINTING
• Glass slide/ membrane/ silicon chip used & uniformly coated with chemical that
interacts with probe and immobilizes it.
• Chemicals used – poly lysine, amino silanes
• Probe selection depends upon use of envisaged for the array.
• Probes usually obtained from cDNA library or PCR amplification of specific DNA
sequences.
• Post processing of Slide
Exposure of slides to UV rays or baking at 80 °C
DNA rendered into single strand
19. 2. SAMPLE PREPARATION AND LABELLING
• Using phenol-based
method
• Using commercially
available kits
• Involves Reverse
transcription step
• Can be taken from libraries
of cDNA
• Crucial step at microarray process
• Usually, fluorochromes are used
• Cy 3- excited by green laser
(normal)
• Cy 5- excited by red laser
(abnormal)
20. Methods
of
labelling
Direct
Other
methods
Indirect
Fig 13 : Direct Labelling Fig 14: Indirect Labelling
Image source -
https://d3i71xaburhd42.net/483cc5f107a4c16e2d240
5dc58dfc741ed1/25-Figure4-1.png
Image source -
https://d3i71xaburhd42.16e2d2405dc58df7b19fc74
1ed1/25-Figure4-1.png
21. 3. HYBRIDIZATION
• Heteroduplexes are formed by base pairing of the probe and the
labelled target sample.
• Blocking before hybridization is done to prevent non – specific
interactions.
Example:
Mixture of succinic anhydride, 1,2-dimethylpyrolidine & sodium borate
• Common components of hybridization solution contains salts,
detergents and buffering agents.
• Optimum temperature is between 45-65 ° C
• It can be manual or robotic.
22. Hybridization
Fig 15 : Hybridization
Fig 16: Types of Hybridization
Manual Robotic
Image source – Stekel , D. (2003)
Image source – Stekel , D. (2003)
23. 4. WASHING
• Reasons for washing are
1. To remove excess hybridization solution and unhybridized sample
2. To increase stringency by reducing cross-hybridization
• Achieved by low salt wash or high temperature wash.
• The automated hybridization stations include washing cycle as a part
of automated process
24. 5. SCANNING
• Scanner is a device that reads the array surface with the help of
fluorescence.
• It uses a light source to excite fluorophores present on the sample,
then detects the emitted light and stores as 16 bit tiff.
Fig 17: Working of scanner
https://www.biocompare.com/Lab-Equipment/20088-Microarray-Scanners-Microarray-Scanner-Systems
25. Scanner Types
Laser Scanners
CCD Scanners
02
01
• CCD is the source of
excitation.
• These capture multiple
images and then switch
altogether creating a more
accurate single image.
• Two different
monochromator is
used.
• PMT detects and
amplifies weak
signals.
Image source - https://www.olympus-global.com/en/news/2002tptb/image/nr02010e.gif
Image source – Stekel , D. (2003)
Fig 18: Laser scanner
working
Fig 19: CCD scanner working
26. IMAGE ANALYSIS AND DATA VISUALIZATION
Image acquisition depends upon a no. of factors such as follows:-
35. Advantages
Disadvantages
DISADVANTAGE
Very expensive
Technical limitations
Resolution of scanner used must be high
Too complex to interpret
Less shelf life of microarrays
ADVANTAGES
.
Provides data or more than thousands of
gene in a small array
Very fast & time saving
Very user-friendly technique
Almost completely computer based
36. Gene expression analysis
TRANSCRIPTOME PROFILING
• Measurements of absolute levels of genes
• Helps to study dynamic changes in gene.
Diagnostic arrays
• Cancer, genetic disorder, infectious diseases
• Rapid, accurate, reliable
Proteomics
• Protein microarrays designed to determine
their interactions and activities on large
scale.
Drug discovery
• Very extensive pharmacological application
• Comaparative analysis of normal cell and
diseased cell for identification of
biochemical constitution of protein .
Toxicology researches
• Study of impact of toxins on cells and next
progeny
Glycomics
• Carbohydrate microarrays developed for
study of interactions and binding of
saccharides.
Crop improvement
• Rice, maize, soybean, sorghum genome have
been sequenced and analysed through this
technique.
• BarleyBase, NSF Rice array, RiceArrayNet, etc.
are databases used for crop improvement
Gene Mutations
• Done by SNPs detection in microarray
APPLICATIONS
37. REFERENCES
1 . C a u s t o n H C , Q u a c ke n b u s h J a n d B r a z m a A ( 2 0 0 3 )
“ M i c r o a r ra y - G e n e E x p r e s s i o n & a n a l y s i s ” 3 - 6 9
2 . S t e ke l D ( 2 0 0 4 ) “ M i c r o a r r a y B i o i n f o r m a t i c s ” C a m b r i d g e
U n i v e r s i t y P r e s s , 1 - 6 0
3 . A p p a s a n i K ( 2 0 0 7 ) “ B i o a r r a y s ” H u m a n a P r e s s , 1 4 8 - 1 7 5
4 . M u l l e r U R a n d N i c o l a u D V ( 2 0 0 4 ) “ M i c r o a r ra y
Te c h n o l o g y a n d i t s a p p l i c a t i o n ” 3 - 8 7
5 . H e l l e r M J ( 2 0 0 2 ) “ D N A M i c r o a r ra y Te c h n o l o g y :
D e v i c e s s y s t e m s & a p p l i c a t i o n s ” 1 2 0 - 1 4 3
6 . F e i z i T, Fa i z o F, C h a i W a n d Wo n g C H ( 2 0 0 3 ) “ C u r r e n t
o p i n i o n i n s t r u c t u ra l b i o l o g y ” 6 3 9 - 6 4 5
