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CourseTeacher
Dr. Shamalamma S.
Professor
Dept, of Plant Biotechnology
DNA MICROARRA
Presented By
Mahendra Reddy
Jn.M.Sc. Plant Biotechnology
PALB5270
 INTRODUCTION
 HISTORY
 PRINCIPLE
 SCANNERS
 EXPERIMENT
 TYPES
 APPLICATIONS
 ANIMATED CLIP
 Microarray” has become a general term, there
are many types now
 DNA microarrays
 Protein microarrays
 Transfection microarrays
 Antibody microarray
 Tissue microarray
 Chemical compound microarray
 …
 We’ll be discussing DNA microarrays
 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.
Historical background :
• Southern blotting was developed in the year 1975.
Sir Edwin Southern
• 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.
Sir Steve Fodor
Sir Patrick
Brown
• Mark schena was proclaimed as the
“Father of Microarray Technology”.
Mark Schena
 The use of a collection of
distinct DNAs in arrays for
expression profiling was first
described in 1987.
 The use of miniaturized
microarrays for gene
expression profiling was first
reported in 1995, and a
complete eukaryotic genome
(Saccharomyces cerevisiae) on
a microarray was published in
1997.
 The core principle behind microarrays is
hybridization.
 Samples are labeled 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
labeling
Hybridisatio
n
Washing
Image acquisition
and
Data analysis
 Fluorescently labeled target
sequences that bind to a
probe sequence generate a
signal.
 The signal depends on.
 The hybridization conditions,
ex: temperature
 washing after hybridization
 Total strength of the signal,
depends upon the; amount
of target sample.
 Laser scanners
 Excellent spatial resolution
 Good sensitivity, but can
bleach fluorochromes
 Still rather slow
 CCD scanners
 Low resolution
 Sensitivity, easily adjustable
(exposure time)
 Faster and cheaper than lasers
 In all cases, raw data are
images showing fluorescence
on surface of chip
 Spotted DNA arrays (“cDNA arrays”)
 Developed by Pat Brown (Stanford)
 PCR products (or long oligos) from known genes (~100 nt) spotted on
glass, plastic, or nylon support.
 Customizable and off the shelf.
 Gene Chips
 Oligonucleotide arrays (Affymetrix)
▪ Small number of 20-25mers/gene
▪ Enabled by photolithography from the
computer industry
▪ Off the shelf
 Ink-jet microarrays (Agilent)
▪ Large number of 25-60mers “printed” directly on glass
▪ Four cartridges: A, C, G, andT
▪ Flexible, rapid, but expensive
 In spotted microarrays, the probes are
oligonucleotides, cDNA or small fragments of
PCR products that correspond to mRNAs.There
probes are synthesized prior to deposition on
the array surface and are then "spotted" onto
glass.
 A common approach utilizes an array of fine
pins or needles controlled by a robotic arm that
is dipped into wells containing DNA probes and
then depositing each probe at designated
locations on the array surface.
 The resulting "grid" of probes represents the
nucleic acid profiles of the prepared probes and
is ready to receive cDNA derived from
experimental or clinical samples.
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
PCR amplification of
target DNA
(cDNA or portion of genomic
DNA)
Consolidate
into plates
Spot as microarray
on glass slides
In oligonucleotide microarrays, the probes are short
sequences designed to match parts of the sequence
of known or predicted open reading frames.
 Although oligonucleotide probes are often used in
"spotted" microarrays, the term "oligonucleotide
array" most often refers to a specific technique of
manufacturing.
 Oligonucleotide arrays are produced by printing
short oligonucleotide sequences designed to
represent a single gene by synthesizing this
sequence directly onto the array surface instead of
depositing intact sequences.
 Sequences may be longer (60-mer probes such as the
Agilent design) or shorter (25-mer probes produced by
Affymetrix) depending on the desired purpose; longer
probes are more specific to individual target genes,
shorter probes may be spotted in higher density
across the array and are cheaper to manufacture.
 One technique used to produce oligonucleotide arrays
include photolithographic synthesis (Agilent and
Affymetrix) on a silica substrate where light and light-
sensitive masking agents are used to "build" a
sequence one nucleotide at a time across the entire
array.
Spotted Arrays
 Relative cheap to make (~$10
slide)
 Flexible - spot anything you
want
 Cheap so can repeat
experiments many times
 Highly variable spot
deposition
 Usually have to make your
own
Affy Gene Chips
 Expensive ($500 or more)
 Limited types avail, no chance
of specialized chips
 Fewer repeated experiments
usually
 More uniform DNA features
 Can buy off the shelf
Experiment
The two samples to be compared
(pairwise comparison) are
grown/acquired.
RNA
DNA
DNA/RNA bound to a protein
The purified RNA is analysed for quality (by capillary
electrophoresis) and quantity (by using a nanodrop
spectrometer)
Optional PCR Amplification
The label is added either in the
RT step or in an additional step
after amplification if present
The labeled samples are then mixed with a propriety
hybridization solution. SDS, SSC, dextran sulfate, a
blocking agent, Denhardt's solution and formamine.
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
Gene expression analysis
 The process of measuring gene expression via cDNA
is called expression analysis.
 Not all the genes in the human genome are active at
all times.
 Used to detect DNA , or detect RNA that may or
may not be translated into proteins.
 Thousand genes are simultaneously assessed.
 Study the effects of certain treatments, diseases,
and developmental stages on gene expression.
 E.g.: identify genes expression changes due to
pathogens or other organisms by comparing
with uninfected cells or tissues.
 Disease diagnosis
Help to investigate about different diseases
 E.g.: Earlier cancers classified on the basis of the
organs in which the tumors develop.
 Now, classify the types of cancer on the basis of
the patterns of gene activity in the
tumor cells.
Extensive application in Pharmacogenomics
 Comparative analysis of the genes.
 Help the identification of the specific proteins
produce by diseased cells.
 Information used to synthesize drugs which
combat with these proteins and reduce their
effect.
 Help to produce very effective
drugs.
 A rapid platform for the research of the
impact of toxins on the cells and their passing
on to the progeny.
 Important forToxicogenomic studies
 Small microarrays to check IDs of organisms
in food and feed (like GMO) and mycoplasms
in cell culture
 Mostly combining PCR and microarray
technology
Study variations in the genes related to the
influence of diets.
 These variations, known as single nucleotide
polymorphism.
 E.g.: Studies are followed to reveal,
 Effects of calorie restriction on gene expression.
 Obesity and high-fat diets.
 Genes responds to gluten and soy protein.
 Provides data for thousands of genes.
 One experiment instead of many.
 Fast and easy to obtain results.
 Huge step closer to discovering cures for
diseases and cancer.
 Different parts of DNA can be used to study
gene expression.
 Very little knowledge is available about many
genes
 Just because mRNA is "turned on" doesn't
mean proteins are made
 The findings may lead to unethical medical
procedures
 Scientists have no standardized way to share
results.
DNA Microarrays are one of the most
effective invention ever developed. A DNA Microarray
is a test that allows for the comparison of thousands of
genes at once. Microarray technology uses chips with
attached DNA sequences as probes for gene
expression. Any DNA in the sample that is
complementary to a probe sequence will become
bound to the chip. Microarray technology is most
powerful when it used on species with a sequenced
genome. The microarray chip can hold sequences from
every gene in the entire genome and the expression of
every gene can be studied simultaneously. Gene
expression data can provide information on the
function of previously uncharacterized genes.
 http://www.gene-chips.com/
 http://www.bio.davidson.edu/courses/genomics/chip/ch
ip.html
 http://www.cs.washington.edu/homes/jbuhler/research/
array
 Dubey C. R, 2008, DNA Chips, A textbook for
Biotechnology, S. Chand and Company Ltd., New Delhi,
13th Edition, Pg. 194 – 197.
 www.gene-chips.com
 www.biotechnology4u.com
 www.biotechnologyforums.com
 www.ehow.com
DNA microarray

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DNA microarray

  • 1. CourseTeacher Dr. Shamalamma S. Professor Dept, of Plant Biotechnology DNA MICROARRA Presented By Mahendra Reddy Jn.M.Sc. Plant Biotechnology PALB5270
  • 2.  INTRODUCTION  HISTORY  PRINCIPLE  SCANNERS  EXPERIMENT  TYPES  APPLICATIONS  ANIMATED CLIP
  • 3.  Microarray” has become a general term, there are many types now  DNA microarrays  Protein microarrays  Transfection microarrays  Antibody microarray  Tissue microarray  Chemical compound microarray  …  We’ll be discussing DNA microarrays
  • 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 : • Southern blotting was developed in the year 1975. Sir Edwin Southern • 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. Sir Steve Fodor Sir Patrick Brown • Mark schena was proclaimed as the “Father of Microarray Technology”. Mark Schena
  • 6.  The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987.  The use of miniaturized microarrays for gene expression profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997.
  • 7.  The core principle behind microarrays is hybridization.  Samples are labeled 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 labeling Hybridisatio n Washing Image acquisition and Data analysis
  • 8.
  • 9.  Fluorescently labeled target sequences that bind to a probe sequence generate a signal.  The signal depends on.  The hybridization conditions, ex: temperature  washing after hybridization  Total strength of the signal, depends upon the; amount of target sample.
  • 10.  Laser scanners  Excellent spatial resolution  Good sensitivity, but can bleach fluorochromes  Still rather slow  CCD scanners  Low resolution  Sensitivity, easily adjustable (exposure time)  Faster and cheaper than lasers  In all cases, raw data are images showing fluorescence on surface of chip
  • 11.  Spotted DNA arrays (“cDNA arrays”)  Developed by Pat Brown (Stanford)  PCR products (or long oligos) from known genes (~100 nt) spotted on glass, plastic, or nylon support.  Customizable and off the shelf.  Gene Chips  Oligonucleotide arrays (Affymetrix) ▪ Small number of 20-25mers/gene ▪ Enabled by photolithography from the computer industry ▪ Off the shelf  Ink-jet microarrays (Agilent) ▪ Large number of 25-60mers “printed” directly on glass ▪ Four cartridges: A, C, G, andT ▪ Flexible, rapid, but expensive
  • 12.  In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs.There probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass.  A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface.  The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive cDNA derived from experimental or clinical samples.
  • 13. Arrayed Library (96 or 384-well plates of bacterial glycerol stocks) PCR amplification of target DNA (cDNA or portion of genomic DNA) Consolidate into plates Spot as microarray on glass slides
  • 14.
  • 15. In oligonucleotide microarrays, the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames.  Although oligonucleotide probes are often used in "spotted" microarrays, the term "oligonucleotide array" most often refers to a specific technique of manufacturing.  Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene by synthesizing this sequence directly onto the array surface instead of depositing intact sequences.
  • 16.  Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer probes produced by Affymetrix) depending on the desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture.  One technique used to produce oligonucleotide arrays include photolithographic synthesis (Agilent and Affymetrix) on a silica substrate where light and light- sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array.
  • 17.
  • 18.
  • 19. Spotted Arrays  Relative cheap to make (~$10 slide)  Flexible - spot anything you want  Cheap so can repeat experiments many times  Highly variable spot deposition  Usually have to make your own Affy Gene Chips  Expensive ($500 or more)  Limited types avail, no chance of specialized chips  Fewer repeated experiments usually  More uniform DNA features  Can buy off the shelf
  • 20.
  • 22. The two samples to be compared (pairwise comparison) are grown/acquired. RNA DNA DNA/RNA bound to a protein The purified RNA is analysed for quality (by capillary electrophoresis) and quantity (by using a nanodrop spectrometer)
  • 23. Optional PCR Amplification The label is added either in the RT step or in an additional step after amplification if present The labeled samples are then mixed with a propriety hybridization solution. SDS, SSC, dextran sulfate, a blocking agent, Denhardt's solution and formamine.
  • 24. 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
  • 25. Gene expression analysis  The process of measuring gene expression via cDNA is called expression analysis.  Not all the genes in the human genome are active at all times.  Used to detect DNA , or detect RNA that may or may not be translated into proteins.  Thousand genes are simultaneously assessed.  Study the effects of certain treatments, diseases, and developmental stages on gene expression.
  • 26.  E.g.: identify genes expression changes due to pathogens or other organisms by comparing with uninfected cells or tissues.  Disease diagnosis Help to investigate about different diseases  E.g.: Earlier cancers classified on the basis of the organs in which the tumors develop.  Now, classify the types of cancer on the basis of the patterns of gene activity in the tumor cells.
  • 27. Extensive application in Pharmacogenomics  Comparative analysis of the genes.  Help the identification of the specific proteins produce by diseased cells.  Information used to synthesize drugs which combat with these proteins and reduce their effect.  Help to produce very effective drugs.
  • 28.  A rapid platform for the research of the impact of toxins on the cells and their passing on to the progeny.  Important forToxicogenomic studies
  • 29.  Small microarrays to check IDs of organisms in food and feed (like GMO) and mycoplasms in cell culture  Mostly combining PCR and microarray technology
  • 30. Study variations in the genes related to the influence of diets.  These variations, known as single nucleotide polymorphism.  E.g.: Studies are followed to reveal,  Effects of calorie restriction on gene expression.  Obesity and high-fat diets.  Genes responds to gluten and soy protein.
  • 31.  Provides data for thousands of genes.  One experiment instead of many.  Fast and easy to obtain results.  Huge step closer to discovering cures for diseases and cancer.  Different parts of DNA can be used to study gene expression.
  • 32.  Very little knowledge is available about many genes  Just because mRNA is "turned on" doesn't mean proteins are made  The findings may lead to unethical medical procedures  Scientists have no standardized way to share results.
  • 33. DNA Microarrays are one of the most effective invention ever developed. A DNA Microarray is a test that allows for the comparison of thousands of genes at once. Microarray technology uses chips with attached DNA sequences as probes for gene expression. Any DNA in the sample that is complementary to a probe sequence will become bound to the chip. Microarray technology is most powerful when it used on species with a sequenced genome. The microarray chip can hold sequences from every gene in the entire genome and the expression of every gene can be studied simultaneously. Gene expression data can provide information on the function of previously uncharacterized genes.
  • 34.
  • 35.  http://www.gene-chips.com/  http://www.bio.davidson.edu/courses/genomics/chip/ch ip.html  http://www.cs.washington.edu/homes/jbuhler/research/ array  Dubey C. R, 2008, DNA Chips, A textbook for Biotechnology, S. Chand and Company Ltd., New Delhi, 13th Edition, Pg. 194 – 197.  www.gene-chips.com  www.biotechnology4u.com  www.biotechnologyforums.com  www.ehow.com