Agriculture Reference
In-Depth Information
Gene expression
Community composition
Genotype analysis
Primary gene information:
genome sequence
cDNA library (ESTs)
environmental library designed with specific primers
environmental isolates
genetic variants
database-derived information (i.e. RDP, GeneBank)
Preparation of nucleic acid for oligonucleotide or gene array:
primer or oligonucleotide design, cloning,
PCR, quality control
Slide and coating selection
Array construction
(i) oligonucleotide array with Affymetrix-type photolithographic
synthesis of oligonucleotides on silica wafer
(ii) oligonucleotide array with polyacrylamide-coated microchip
(iii) gene array with commercially available, or self-built robot
Growth of cells/collection of sample
RNA/DNA extraction and purification
Fluorescent labelling of nucleic acid
- reverse transcriptase
- Klenow DNA polymerase
- direct chemical labelling of RNA or DNA
Hybridize with two-colour fluorescently labelled samples
Visualize with
- confocal scanning laser
- CCD camera optimized to detect fluorescent signals
Data processing with commercially available software
or freeware
Statistical analysis: correlation analysis, cluster analysis
pattern recognition
Fig. 6.5.
Flowchart for DNA microarray experimental strategy. Results from a section of an
actual array experiment are shown in which genes expressed under denitrifying conditions are
compared with their expression under aerobic conditions. The array image has been converted
from colour (red versus green), which is much more easily quantified, to black and white. Nonethe-
less, differences in expression can be seen. The microarray was constructed with PCR products
designed from the Shewanellaoneidensisstrain MR-1 genome sequence. This organism is of
interest because it can use a variety of electron acceptors for growth including Fe
3
+
, Mn
4
+
, NO
3
−
and O
2
.
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