82
M.S. Clark et al.
(Faure et al. 2008). In a variant of this technique (called ecoTILLING), rather than
using a particular marker to follow a gene, the researcher begins with a gene (usually selected based on genome sequence) which is potentially related to a phenotype
of interest (Comai et al. 2004). The ecoTILLING method allows the rapid detection of natural sequence variants of this gene within a population of genotypes
by PCR amplification of a population of alleles followed by mismatch detection
using the CEL1 or ENDO1 endonucleases, which cleave DNA at polymorphic sites
(Comai et al. 2004). Variant alleles can then be followed using molecular markers
and associated with particular traits of adaptive significance.
3.1.7 DNA Studies: Barcoding
The development of rapid sequencing methods including new developments based
on pyro-sequencing technology may also be applied to barcoding approaches.
Barcoding is a populist name given to the technique of assigning a unique identifier to every known species. This technique is not only useful for population and
taxonomy studies, but also marine forensic analyses. However, the application of
this technology has already been comprehensively explained in Chapter 1and will
not be explored further here, as the emphasis will remain on large-scale population
analyses.
3.1.8 RNA Studies: Microarrays or Gene Chips
Microarrays represent a method of analysing the expression of many genes at the
same time. Thousands of sequences are individually attached (spotted) to a small
glass slide (typically the size of a microscope slide) with each spot on the slide representing a gene sequence. These sequences can either be cDNAs or oligonucleotides
designed using EST or genomic sequence information. Hybridization of samples
to the microarray can be performed using either a single or double fluorescent dye
system, the choice of which depends on the technology used to manufacture the
chip (cf. Gibson 2002). Statistical analyses are then applied to the colour and level
of intensity of fluorescence to determine how the applied treatment has affected
gene expression. Generally genes that show more than a 2× increase in fluorescence under any conditions are regarded as having significantly changed under the
assumption that the signal is normally distributed between replicates within the
two samples under scrutiny. Whilst this is clearly a powerful technique for mass
screening of gene expression, experiments have to be carefully designed with rigorous consideration of what constitutes a “control” and planning sample replications.
If too many different variables are introduced into the experiments, then the data
can become too noisy and no meaningful information can be extracted. Currently
it is essential to validate microarray results using other methods of transcriptional
analysis, such as Q-PCR.
M.S. Clark et al.
(Faure et al. 2008). In a variant of this technique (called ecoTILLING), rather than
using a particular marker to follow a gene, the researcher begins with a gene (usually selected based on genome sequence) which is potentially related to a phenotype
of interest (Comai et al. 2004). The ecoTILLING method allows the rapid detection of natural sequence variants of this gene within a population of genotypes
by PCR amplification of a population of alleles followed by mismatch detection
using the CEL1 or ENDO1 endonucleases, which cleave DNA at polymorphic sites
(Comai et al. 2004). Variant alleles can then be followed using molecular markers
and associated with particular traits of adaptive significance.
3.1.7 DNA Studies: Barcoding
The development of rapid sequencing methods including new developments based
on pyro-sequencing technology may also be applied to barcoding approaches.
Barcoding is a populist name given to the technique of assigning a unique identifier to every known species. This technique is not only useful for population and
taxonomy studies, but also marine forensic analyses. However, the application of
this technology has already been comprehensively explained in Chapter 1and will
not be explored further here, as the emphasis will remain on large-scale population
analyses.
3.1.8 RNA Studies: Microarrays or Gene Chips
Microarrays represent a method of analysing the expression of many genes at the
same time. Thousands of sequences are individually attached (spotted) to a small
glass slide (typically the size of a microscope slide) with each spot on the slide representing a gene sequence. These sequences can either be cDNAs or oligonucleotides
designed using EST or genomic sequence information. Hybridization of samples
to the microarray can be performed using either a single or double fluorescent dye
system, the choice of which depends on the technology used to manufacture the
chip (cf. Gibson 2002). Statistical analyses are then applied to the colour and level
of intensity of fluorescence to determine how the applied treatment has affected
gene expression. Generally genes that show more than a 2× increase in fluorescence under any conditions are regarded as having significantly changed under the
assumption that the signal is normally distributed between replicates within the
two samples under scrutiny. Whilst this is clearly a powerful technique for mass
screening of gene expression, experiments have to be carefully designed with rigorous consideration of what constitutes a “control” and planning sample replications.
If too many different variables are introduced into the experiments, then the data
can become too noisy and no meaningful information can be extracted. Currently
it is essential to validate microarray results using other methods of transcriptional
analysis, such as Q-PCR.