This document outlines the principles and applications of DNA microarrays, which are used to measure gene expression and genetic variations by hybridizing DNA sequences on a solid surface. It discusses different types of microarrays, including cDNA and oligonucleotide microarrays, and their historical development from Southern and Northern blotting techniques. The document also highlights various applications such as gene expression profiling, SNP analysis, and comparative genomic hybridization.

1. : Ujjwal Sirohi
Phd research scholar
Microarray
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2.  This is an old ppt of mine
 Reliable information
 Concern with other materials too

3.  A DNA microarray (also commonly known as DNA chip or
biochip) is a collection of microscopic DNA spots attached to a
solid surface.
 Scientists use DNA microarray to measure the expression level of
genes simuntaneously or to genotype multiple regions of a
genome.
 Each DNA spot contains picomoles os aspecific DNA aequence
such as probes(or reporters of oligos).
 These can be a short section of a gene or other DNA element that
are used to hybridise a cDNA or cRNA (also called anti-sence
RNA) sample (called target) under high-stringency conditions.

4.  Probe target hybridisation is usually detected and quantified by
detection of fluorophore-, silver-, or chemiluminescence –labelled
targets to determine relative advance of nucleic acid sequence in
the targets.
 Microarrays are based on the ability of complementary strands of
DNA (or DNA and RNA) to hybridize to one another in solution
with high specificity.
 There are now many variations.We’ll take a quick look at the two
basic types:Affymetrix (high density oligonucleotide) and glass
slide (cDNA, long oligo, etc). Both are conceptually similar, with
differences in manufacture and details of design and analysis.

5. principle
 The core principle behind microarray is hybridisation b/w two
DNA strands, the property of complementary nucleic acid
sequences to specifically pair with each other by forming hydrogen
bonds b/w complementary nucleotide base pair.
 A high number of complementary base pair in a nucleotide
sequence means tighter non-covalent bonding between the two
strands.
 After washing off non-specific bonding sequences, only strong
paired strands will remain hybridised.
 Fluorescently labeled target sequences that bind to a probe
sequence generate a signal that depends on the hybridisation
condition(such as temperature), and washing after hybridisation.

6.  Total strength of the signal from the spot(feature), depends
upon the amount of target sample binding to the probes
present on that spot.
 Microarrays use relative quantitation in which the intensity of
a feature is compaired to the intensity of the same feature
under a different condition, and the identity of the feature is
known by its position.

7. Types of micro array includes
 DNA microarrays, such as cDNA microarrays and oligonucleotide
microarrays, SNPs, CHiP
 MMChips, for surveillance of microRNA populations
 Protein microarrays (protein-protein interactions)
 Tissue microarrays
 Cellular microarrays (also called transfection microarrays
 Chemical compound microarrays
 Antibody microarrays (proteomics)
 Carbohydrate arrays (glycoarrays)

8. History of DNA microarray
 Microarrays descend from Southern and Northern
blotting. Unknown DNA is transferred to a membrane
and then probed with a known DNA sequence with a
label
 In Microarrays, the known DNA sequence (or probe) is
on the membrane while the unknown labeled DNA (or
target) is hybridized and then washed off so only specific
hybrids remain.
 Dot Blots of different genes in an array were used to
assay gene expression as early as 1987.
 Complete genome of all Saccharomyces cerevisiae ORFs on
a microarray published in 1997 by Lashkari et al.

9. what can be measured using microarrays?
Amount of mRNA expressed by a gene.
gene expression array, exon array, tiling array
2. Amount of mRNA expressed by an exon.
exon array, tiling array
3. Amount of RNA expressed by a region of DNA.
tiling array
4. Which strand of DNA is expressed.
exon array, tiling array
5. Which of several similar DNA sequences is present in the
genome.
SNP array
6 . How many copies of a gene is present in the genome.
gene expression array, exon array, tiling array
7. Where a known protein has bound to the DNA. (ChIP on chip)
promoter array, tiling array

10. Basics of microarray
 DNA attached to solid
support
 Glass, plastic, or nylon
 RNA is labeled
 Usually indirectly
 Bound DNA is the
probe
 Labeled RNA is the
“target”

11. Slide based microarray
 For slide-based microarrays, the probe DNA is affixed
directly to the surface of a glass microscope slide.The probe
DNA can range from a medium-length oligonucleotide (e.g.,
60 nt) to an entire cDNA clone or larger. Oligonucleotide
arrays have become more common and can be obtained from
several different commercial vendors.
 The DNA is deposited on the slide by any of a number of
methods, including “printing” with what is essentially an ink-
jet printer and spotting using a robotically controlled set of
fine-tipped metal pins.An example of the latter is seen in the
following slides:

12. DNA spotting I
 DNA spotting usually uses
multiple pins
 DNA in microtiter plate
 DNA usually PCR amplified
 Oligonucleotides can also be
spotted

13. Commertial DNA spotter

14. Slide based microarrays
 In general, slide-based arrays are used to make a direct
comparison between two different RNA samples.These can be
a tissue sample vs. a reference, mutant vs. wild type, treated
vs. control, etc.The microarray provides a readout of the
relative differences in abundance of the RNAs present in each
sample.
extract
mRNA
make
labeled
cDNA
hybridize to
microarray
more in “A”
more in “B”
equal in A & B
cell type B
cell type A

15. cDNA microarrays: key points
• hybridize two samples/chip (i.e., direct comparison of
samples)
• non-standardized production can affect reproducibility (i.e.,
depends a lot on who made them), although there are now
many quality-controlled commercial arrays available
• longer sequences can have cross-hybridization with other
genes
• don’t necessarily need to know all genes in genome: can use
unsequenced ESTs, for instance

16. Affymatrix gene chips
 Oligonucleotides
 Usually 20–25 bases in length
 10–20 different oligonucleotides for each gene
 Oligonucleotides for each gene selected by computer
program to be the following:
 Unique in genome
 Nonoverlapping
 Composition based on design rules
 Empirically derived

17. How microarrays are made: Affymatrix
gene chips
 Oligonucleotides synthesized on silicon chip
 One base at a time
 Uses process of photolithography
 Developed for printing computer circuits

18. Affymatrix gene chips
 Each gene is represented by a “probe set” consisting of 12-20
probes of 25 nt each. Each probe has a corresponding
“mismatch” probe with a single base difference at the 13th
nucleotide. Labeled RNA is hybridized to the array, and a
measure of abundance is calculated based on the amount of
hybridization seen for the entire probe set, correcting for
hybridization to the mismatch probes, which indicates
possible non-specific effects.
probe pair Mismatch probe cells

19. probe pair Mismatch probe cells
(12-20/gene)
Affymetrix GeneChips

20. Affymatrix :key points
• can hybridize only one sample/chip (i.e., no direct
comparisons of 2 samples)
• standardized production tends to give good reproducibility
• limited amount of probe sequence can be problematic (miss
alternative splices, bias toward one end of transcript,
dependent on good genome annotation), but can also be
helpful in limiting cross- hybridization

21. Comparison of microarray hybridisation
 Spotted microarrays
 Competitive hybridization
 Two labeled cDNAs hybridized to same slide
 Affymetrix GeneChips
 One labeled RNA population per chip
 Comparison made between hybridization intensities of same
oligonucleotides on different chips

22. Uses of microarray
 Gene expression profiling
- Monitoring expression levels for thousands of genes simultaneously.
Three other common applications:
 Array CGH (Comparative genomic hybridization)
-Assessing genome content in different cells or closely related
organisms.
 SNP array (single nucleotide polymorphism)
- Identifying single nucleotide polymorphism among alleles within or
between populations.
 ChIP-on-chip (Chromatin immunoprecipitation)
- Determining protein binding site occupancy throughout the genome.
 Methylation arrays (immonoprecipitate methylated DNA_
-Determining which regions of DNA are methylated to determine
epigenetics

23. Thank you