DNA microarray
is a hybridization technique performed in microscopic glass slide with surface
modification, anchoring specific set of probes sequences complementary to the
Target. Upon complementation with the target sequence fluorescence signal can
be captured and state of the test sample can be identified.
is a hybridization technique performed in microscopic glass slide with surface
modification, anchoring specific set of probes sequences complementary to the
Target. Upon complementation with the target sequence fluorescence signal can
be captured and state of the test sample can be identified.
DNA
microarrays can be used to measure changes in expression levels, to
detect single nucleotide polymorphisms (SNPs), or to genotype or
targeted resequencing.
microarrays can be used to measure changes in expression levels, to
detect single nucleotide polymorphisms (SNPs), or to genotype or
targeted resequencing.
Principle of DNA Microarray
The
principle behind microarray is the hybridization between two DNA strands.
Complementary sequence of the DNA pair specifically by forming hydrogen bonds,
the more number of complementary base pairs in a nucleotide sequence will have
a tighter non-covalent bonding between the two strands.
principle behind microarray is the hybridization between two DNA strands.
Complementary sequence of the DNA pair specifically by forming hydrogen bonds,
the more number of complementary base pairs in a nucleotide sequence will have
a tighter non-covalent bonding between the two strands.
After the
hybridization of target DNA to the probes attached on the glass slide, slides
are washed to remove any non-specifically bound sequences, only strongly paired
strands will remain hybridized.
hybridization of target DNA to the probes attached on the glass slide, slides
are washed to remove any non-specifically bound sequences, only strongly paired
strands will remain hybridized.
Fluorescently
labelled target sequences that bind to a probe sequence generate a signal that
depends on the hybridization conditions (such as temperature), and washing
after hybridization. Total strength of the signal, from a 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 compared to the intensity of the same feature under a different condition,
and the identity of the feature is known by its position.
labelled target sequences that bind to a probe sequence generate a signal that
depends on the hybridization conditions (such as temperature), and washing
after hybridization. Total strength of the signal, from a 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 compared to the intensity of the same feature under a different condition,
and the identity of the feature is known by its position.
Preparing Glass Slide for Surface Modification
Glass slide used for surface modification microarray need to be clean, to remove any organic contaminats and dirt glass can be cleaned with Ethanol, Toulene or Etching with pirhana. Etching with pirhana (7:3 H2SO4:H2O2) is the better choice among the other cleaning methods. One need to be careful while while handling pirhana solution as it is a strong oxidizer.
Surface Modification Strategies
Various Surface modification are developed for DNA microarray, few of the most commonly used ones are
- Poly – L- Lysine,
- 3-aminopropyltrimethoxysilane (APS),
- 3-glycidoxypropyltrimethoxysilane (GPS) and
- Aldehyde or carboxylic acid.
Oligo attachment to the modified glass surface
- Oligo attachment to PLL coated glass slide
 |
|
- Oligo attachment to APS coated glass slide
 |
| 3-aminopropyltrimethoxysilane (APS) hydrogen bonding with an oligonucleotide. |
- Oligo attachment to GPS coated glass slide
 |
| 3-glycidoxypropyltrimethoxysilane (GPS) covalently bound to an amine-terminated oligonucleotide. |
|
One-half of a DAB dendrimer hydrogen bonding with an oligonucleotide |
Spotting Oligo Probes on the glass slide
- Mechanical Spotting
- Ink Jetting
Microarray Workflow
Microarray Detection / Capture
List of commercially availbale DNA Microarrays
- ACLARA Bio Sciences
- GeneChipTM – Affymetrix
- LabChip – Caliper Technologies
- LabCD system – Gamera Bioscience
- Genetix Ltd
- Agilent
- Illumina
- Incyte Microarray Systems
- Nanogen
- Sequenom and
- GenoSensor – Vysis Inc. (Downers Grove, IL)
References
Technical Resources, Array It Corp
Technical Resources, Affymetrix
A Beginer’s Guide to DNA microarrays
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