Microarray

1

• Microarray
• -DNA microarray hybridization is a method of
  choice for high-throughput RNA expression
  analysis.

2

• -Its a high throughput technology.
• -In general array is an arrangment of points in
  rows and columns.
• A microarray is an extension of the concept
  having a very small arrangement of many points in
  rows and columns.
• This technique basically originated from Southern
  blotting and in this technique a series of high
  density DNA spots bound to some solid support.
• Microarray bind DNA to a solid base (usually a
  glass), using robot automated processes (with
  high precision).
• The precloned immobilized DNA molecules can be
  probed with labeled complementary sequences of
  DNA.

3

• DNA microarrays have spots that contain known
  sequences of DNA to which labeled sample of
  unknown composition is hybridized and then
  detected.
• There are two major types of DNA array used in
  expression analysis
• 1. Spotted DNA arrays
• 2. Printed DNA arrays

4

• Spotted DNA arrays
• -A spotted DNA array is made by transfering
  (spotting) actual DNA clones or PCR products
  derived therefrom) individually onto a solid
  support such as nitrocellulose membrane in a
  precise grid pattern.
• -Corresponding clones provide positive signal.
• -Comparative gene analysis requires the
  preparation of duplicate arrays or the sequential
  probing, stripping and re-probing of the same
  array with two different probes.
• -Nylon and glass microarrays are available.
• -Glass is an inert substrate and thus coated with
  negatively charged phosphate groups of DNA are
  exploited for immobilization on positively
  charged surface groups provided.
• eg. Poly-L-lysine and subsequently DNA
  cross linked to the surface.
• -Also, amino groups can be attached to DNA and
  immobilized on aldehydes or epoxy-derivatized
  surfaces.

5

• -In case of membrane, probe is usually labeled
  with radioactive or enzymatic, which provide poor
  signal. While fluorescent probes having higher
  resolution, but becoz of autofluorescence of the
  substrate in case of nylon membranes has a high
  auto-fluorescence, generating low signal-to-noise
  ratio.
• -Interestingly, glass is a non-porous substance
  having little auto fluorescence, fluorescent
  probes can be used.
• -Different fluorophores can be used to lable
  different RNA populations.
• eg. Two different probes Cy3 and Cy5 which
  provide bright red and green fluorescence ,
  respectively.
• So, if a particular cDNA is present only in Cy3
  labeled population the spot on the array is
  green and red in case of Cy5, but if present in
  both population contain equal proportion spot
  will appear yellow.

6

• -In case of bacteria and also in Saccharomyces
  cerevisiae, genomic array can be derived becoz
  either lack of introns or few numbers only.
• -Complete c-DNA sequence is not needed only the
  signature sequences or ESTs are sufficient to
  use.
• EST (expressed sequence tags) By using high
  throughput sequencing technology, thousand of
  clones picked randomly from cDNA libraries and
  subjected to single-pass sequencing to generate
  2-300 bp cDNA signatures called expressed
  sequence tags (EST).

7

• -Actually, the spotted arrays were having some
  differences in the features (spots), and to
  overcome this printed technologies were
  developed.
• -Spotting pins, piezoelectric devices similar to
  inkjet printers and bubblejet printheads that
  deposited DNA samples on the substrate as a
  bubble extended from the nozzle.

8

• Oligonucleotide chips principle
• -Sequences are obtained from public or private
  databases and synthesized on the chip.
• -Each gene is reprinted by 20 non-overlapping
  oligonucleotides each with a perfect match (PM)
  and mismatch (MM) feature.

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• Oligonucleotide chips are manufactured by in situ
  oligonucleotide synthesis
• -Affymetrix Inc. developed a light directed
  printing technology known as photolithography.
• -Glass or silicon wafer is hydroxylated and
  silanized so that DNA can be covalently attached
  to the surface in a simple chemical reaction.
• -Covalent binding sites are blocked by a
  photolabile protecting group.
• A chromium mask is then applied to the surface of
  the chip which determines which areas are exposed
  to light.
• Under illumination, the protecting groups in
  these areas are destroyed. Allowing the addition
  of a single nucleotide, which is also blocked
  with a photo labile protecting group.
• This method allows the production of most
  accurate and densest arrays currently available
  up to 64,000 features over an area slightly
  larger than
• 1 cm2 (GeneChip).

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• -In contrast to DNA arrays, oligonucleotide chip
  arrays are not double stranded clones or PCR
  products, but single stranded targets ranging
  from 25-70 nucleotides.
• -Probes for chip hybridization are made from
  cleaved, biotinylated cRNA (RNA that has been
  transcribed in vitro from cDNA).

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• Properties of different types of DNA array for
  expression analysis
• Property Spotted nylon
  spotted glass Affymetrix
• Target composition dsDNA (genomic, cDNA or PCR
  products) ss-oligonucleotides
• Target source derived from RNA, maintained
  clones Seq derived
  from
• 
  
  public or private
• 
  
  databases. Chemically
• 
  
  synthesized
• Target size 100-300bp
  
  20-25nt
• Array format Individual features
  represent non-redundant clones Single clones
  20 non
• 
  
  overlapping oligos
• Density 1-10
  gt5000
  64,000 to 10,00000
• Manufacture Robotized or manual
  robotized On-chip
  photolithographic
• Substrate Nylon
  Glass
  Glass or silicon
• Probe labelling Radioactive or enzymatic
  Dual fluorescent Fluorescent
• Hybridization High vol (50ml)
  10 ul
  200ul
• 65 degree C
  65 degree C 40
  degree C
• Data acquisition Autorad, phosphorimager
  Confocal Confocal