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MICROARRAYS




        Compiled by: Dr. Prabhash
What is Microarray ???

Microarrays are sets of miniaturized chemical reaction areas
that may be used to test DNA fragments, antibodies,
or proteins.
Each reaction area or spot is having immobilised target which
is hybridised with complimentary probe present in the testing
sample.
Various types of microarrays are :
• DNA microarrays, such as cDNA microarrays, oligonucleotide
  microarrays and SNP microarrays
• MMChips, for surveillance of microRNA populations
• Peptide microarrays, for detailed analyses or optimization of
  protein-protein interactions
• Tissue microarrays
• Cellular microarrays (also called transfection microarrays)
• Chemical compound microarrays
• Antibody microarrays
• Carbohydrate arrays (glycoarrays)
• Phenotype microarrays
DNA Microarray
• commonly known as DNA chip.
 -Bio chip is a small rectangular solid surface that is made of glass or
  silicone.
-short DNA or RNA targets are anchored to its surface. (no. may vary
from 10-20 to hundreds of thousands)
- Each known gene occupies a particular “spot” on the chip.
-the DNA sample which is to be analysed is amplified by PCR.
-now sample DNA is labelled with fluorescent tag
-this fluorescent DNA sample is loaded on chip
-Hybridization occurs between sample DNA and target, allowing
thousands of hybridization reactions to occur at the same time.
-complete sequence matching result in bright
fluorescence and even a single base mismatching
result in a dimmer fluorescence signal.
The solid surface of Biochip
• Substrates for arrays are usually silicon chips or glass
  microscope slides.
• Glass is a readily available and inexpensive support medium
  that has a relatively homogeneous chemical surface whose
  properties have been well studied and is amenable to
  chemical modification using very versatile and well developed
  silanization chemistry.
• The two-dimensional surface is typically prepared by treating
  the glass or silicon surface with an amino silane which results
  in a uniform layer of primary amines or epoxides.
• These modifications results in several advantages:
1. the linkages are chemically stable,
2. sufficiently long to eliminate undesired steric interference
    from the support, and
3. hydrophilic enough to be freely soluble in aqueous solution
    and not produce non-specific binding to the support.
• Once these modifications have activated the surface, the
  efficiency of attaching the oligonucleotides depends largely on
  the chemistry used and how the oligonucleotide targets are
  modified.
Fabrication of the surface with oligonucleotide



               • Fabrication is done by two methods:
                       1. Spotted microarray
     2. In situ synthesised array / oligonucleotide microarrays
• Spotted microarray:
    In spotted microarrays, the probes can be
i. oligonucleotides,
ii. cDNA or
iii. small fragments of PCR products that correspond to
      mRNAs.
      The probes are synthesized prior to deposition on the array
surface and are then "spotted" onto glass.




                                      Spotting is done by a technique
                                      which utilises atomic force
                                      microscopy;
                                      known as Dip Pen Nanolithography
• This technique is used by research scientists around the world
  to produce "in-house" printed microarrays from their own
  labs.
• These arrays may be easily customized for each experiment,
  because researchers can choose the probes and printing
  locations on the arrays, synthesize the probes in their own lab
  (or collaborating facility), and spot the arrays.
• This provides a relatively low-cost microarray that may be
  customized for each study, and avoids the costs of purchasing
  often more expensive commercial arrays that may represent
  vast numbers of genes that are not of interest to the
  investigator.
• In-house spotted microarrays may not provide the same level
  of sensitivity compared to commercial oligonucleotide
  arrays, possibly owing to the small batch sizes and reduced
  printing efficiencies when compared to industrial
  manufactures of oligo arrays.
• Oligonucleotide array:
• Oligonucleotide arrays are produced by printing short
  oligonucleotide sequences designed to represent a single
  gene or family of gene splice-variants.
• Printing is done by synthesizing this sequence directly onto
  the array surface instead of depositing intact sequences as
  done in spotted microarray.
• technique used to produce oligonucleotide arrays
  include photolithographic synthesis (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.
• Oligonucleotide array synthesis by Photolithography :
Fluoroscence tagging of DNA sample

• Fluorescent dyes commonly used for cDNA labeling
  include Cy3, which has a fluorescence emission wavelength of
  570 nm (corresponding to the green part of the light
  spectrum), and Cy5 with a fluorescence emission wavelength
  of 670 nm (corresponding to the red part of the light
  spectrum).
Hybridization
Array analysis
• Hybridization is analysed by laser induced fluorescence. (LIF)
Two channel microarray




Relative intensities of each fluorophore may then be used in ratio-based
analysis to identify up-regulated and down-regulated genes.
Single channel microarray
• In single-channel microarrays or one-color microarrays, the
  arrays provide intensity data for each probe or probe set
  indicating a relative level of hybridization with the labelled
  target.
• One strength of the single-dye system lies in the fact that an
  aberrant sample cannot affect the raw data derived from other
  samples, because each array chip is exposed to only one sample
  (as opposed to a two-colour system in which a single low-quality
  sample may drastically impinge on overall data precision even if
  the other sample was of high quality).
• Another benefit is that data are more easily compared to arrays
  from different.
• A drawback to the one-color system is that, when compared to
  the two-colour system, twice as many microarrays are needed to
  compare samples within an experiment.
Application of DNA microarray
Analysis of Gene Expression
• Examining expression during development
  or in different tissues

• Comparing genes expressed in normal vs.
  diseased states

• Analyzing response of cells exposed to
  drugs or different physiological conditions
Monitoring Changes in Genomic DNA

• Identify mutations
• Examining genomic instability such as in certain
  cancers and tumors (gene amplifications,
  translocations, deletions)
• Identifying polymorphisms (SNPs)
• Diagnosis: chips have been designed to detect
  mutations in p53, HIV, and the breast cancer
  gene BRCA-1
Applications in Medicine

• Inferring regulatory networks
• Pathogen analysis (rapid genotyping)
• Staging of leukemias
Applications in Drug Discovery
                Drug Discovery
• Identify appropriate molecular targets for
  therapeutic intervention (small molecule / proteins)
• Monitor changes in gene expression in response
  to drug treatments (up / down regulation)
• Analyze patient populations (SNPs) and response


          Targeted Drug Treatment
• Pharmacogenomics: individualized treatments
• Choosing drugs with the least probable side
  effects
Nucleic acid
                    techniques
  Detection                             Discrimination
 techniques                              techniques
UV absorbance                           electrophoresis

Fluoroscence                             HPLC & mass
   staining                             spectrophotom
                                             etry
                                         Hybridization
                                            assay
                    Solid phase                            Solution phase
                   hybridization                            hybridization
                 Dot-blot and line                             RT-PCR
                   probe assay
                 Arrays (micro and                        PCR melting assay
                 medium density)
                In situ hybridization                          others

                  Southern and
                 northern blotting
references
• http://ebookbrowse.com/gdoc.php?id=253431199&url=4d57
  4becdcfd5331bdfc535282916ca3
• http://www.youtube.com/watch?v=zFnJQjcnhGQ
• http://www.columbia.edu/~bo8/undergraduate_research/pro
  jects/sahil_mehta_project/uses.htm

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Microarray by dr.prabhash

  • 1. MICROARRAYS Compiled by: Dr. Prabhash
  • 2. What is Microarray ??? Microarrays are sets of miniaturized chemical reaction areas that may be used to test DNA fragments, antibodies, or proteins. Each reaction area or spot is having immobilised target which is hybridised with complimentary probe present in the testing sample.
  • 3. Various types of microarrays are : • DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays and SNP microarrays • MMChips, for surveillance of microRNA populations • Peptide microarrays, for detailed analyses or optimization of protein-protein interactions • Tissue microarrays • Cellular microarrays (also called transfection microarrays) • Chemical compound microarrays • Antibody microarrays • Carbohydrate arrays (glycoarrays) • Phenotype microarrays
  • 4. DNA Microarray • commonly known as DNA chip. -Bio chip is a small rectangular solid surface that is made of glass or silicone. -short DNA or RNA targets are anchored to its surface. (no. may vary from 10-20 to hundreds of thousands) - Each known gene occupies a particular “spot” on the chip. -the DNA sample which is to be analysed is amplified by PCR. -now sample DNA is labelled with fluorescent tag -this fluorescent DNA sample is loaded on chip -Hybridization occurs between sample DNA and target, allowing thousands of hybridization reactions to occur at the same time. -complete sequence matching result in bright fluorescence and even a single base mismatching result in a dimmer fluorescence signal.
  • 5.
  • 6. The solid surface of Biochip • Substrates for arrays are usually silicon chips or glass microscope slides. • Glass is a readily available and inexpensive support medium that has a relatively homogeneous chemical surface whose properties have been well studied and is amenable to chemical modification using very versatile and well developed silanization chemistry. • The two-dimensional surface is typically prepared by treating the glass or silicon surface with an amino silane which results in a uniform layer of primary amines or epoxides.
  • 7. • These modifications results in several advantages: 1. the linkages are chemically stable, 2. sufficiently long to eliminate undesired steric interference from the support, and 3. hydrophilic enough to be freely soluble in aqueous solution and not produce non-specific binding to the support. • Once these modifications have activated the surface, the efficiency of attaching the oligonucleotides depends largely on the chemistry used and how the oligonucleotide targets are modified.
  • 8. Fabrication of the surface with oligonucleotide • Fabrication is done by two methods: 1. Spotted microarray 2. In situ synthesised array / oligonucleotide microarrays
  • 9. • Spotted microarray: In spotted microarrays, the probes can be i. oligonucleotides, ii. cDNA or iii. small fragments of PCR products that correspond to mRNAs. The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. Spotting is done by a technique which utilises atomic force microscopy; known as Dip Pen Nanolithography
  • 10. • This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. • These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. • This provides a relatively low-cost microarray that may be customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator. • In-house spotted microarrays may not provide the same level of sensitivity compared to commercial oligonucleotide arrays, possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo arrays.
  • 11. • Oligonucleotide array: • Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants. • Printing is done by synthesizing this sequence directly onto the array surface instead of depositing intact sequences as done in spotted microarray. • technique used to produce oligonucleotide arrays include photolithographic synthesis (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.
  • 12.
  • 13. • Oligonucleotide array synthesis by Photolithography :
  • 14. Fluoroscence tagging of DNA sample • Fluorescent dyes commonly used for cDNA labeling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum).
  • 16. Array analysis • Hybridization is analysed by laser induced fluorescence. (LIF)
  • 17.
  • 18. Two channel microarray Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes.
  • 19. Single channel microarray • In single-channel microarrays or one-color microarrays, the arrays provide intensity data for each probe or probe set indicating a relative level of hybridization with the labelled target. • One strength of the single-dye system lies in the fact that an aberrant sample cannot affect the raw data derived from other samples, because each array chip is exposed to only one sample (as opposed to a two-colour system in which a single low-quality sample may drastically impinge on overall data precision even if the other sample was of high quality). • Another benefit is that data are more easily compared to arrays from different. • A drawback to the one-color system is that, when compared to the two-colour system, twice as many microarrays are needed to compare samples within an experiment.
  • 20. Application of DNA microarray
  • 21. Analysis of Gene Expression • Examining expression during development or in different tissues • Comparing genes expressed in normal vs. diseased states • Analyzing response of cells exposed to drugs or different physiological conditions
  • 22. Monitoring Changes in Genomic DNA • Identify mutations • Examining genomic instability such as in certain cancers and tumors (gene amplifications, translocations, deletions) • Identifying polymorphisms (SNPs) • Diagnosis: chips have been designed to detect mutations in p53, HIV, and the breast cancer gene BRCA-1
  • 23. Applications in Medicine • Inferring regulatory networks • Pathogen analysis (rapid genotyping) • Staging of leukemias
  • 24. Applications in Drug Discovery Drug Discovery • Identify appropriate molecular targets for therapeutic intervention (small molecule / proteins) • Monitor changes in gene expression in response to drug treatments (up / down regulation) • Analyze patient populations (SNPs) and response Targeted Drug Treatment • Pharmacogenomics: individualized treatments • Choosing drugs with the least probable side effects
  • 25.
  • 26.
  • 27. Nucleic acid techniques Detection Discrimination techniques techniques UV absorbance electrophoresis Fluoroscence HPLC & mass staining spectrophotom etry Hybridization assay Solid phase Solution phase hybridization hybridization Dot-blot and line RT-PCR probe assay Arrays (micro and PCR melting assay medium density) In situ hybridization others Southern and northern blotting
  • 28. references • http://ebookbrowse.com/gdoc.php?id=253431199&url=4d57 4becdcfd5331bdfc535282916ca3 • http://www.youtube.com/watch?v=zFnJQjcnhGQ • http://www.columbia.edu/~bo8/undergraduate_research/pro jects/sahil_mehta_project/uses.htm