Chemistry Reference
In-Depth Information
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Tag
Isolate tags
Link tags together and sequence
Quantitate tags and determine the pattern of gene expression
Figure 12.3
In the SAGE methodology, short sequence tags (10-14 bp) obtained from a unique position within
each transcript uniquely identify transcripts. The expression level of the corresponding transcript
is shown by the number of times a particular tag is observed. (Adapted from Varzakas, T. et al.,
in
Tomatoes and Tomato Products, Nutritional, Medicinal and Therapeutic Properties
, edited
by V.R. Preedy and R.R. Watson, pp. 515-536, Science Publishers, Enield, NH, 2008. With
permission.)
12.4.1.4 Microarrays
More recent developments involve the use of DNA microarray technology in altered gene
expressionwithamoreeficientandinformativeway(LockhartandWinzeler2000;Pandaetal.
2003;Mockleretal.2005).UsingDNAmicroarrays,theexpressionofalargenumberofgenescan
beanalyzedsimultaneouslyandinasemiquantitativemanner.Thisallowsfortheanalysisofdiffer-
entmetabolicpathwaysininteractionandfacilitatestheidentiicationofkeyresponsivegenes.For
alimitednumberofspecies,microarraysthatrepresentallidentiiedmetabolicroutesandgenes
active therein have been constructed. These are the so-called whole genome arrays, oligo-arrays
whereallexpressedgenesequencesarerepresentedbyoneormoreshortDNAsequences,usually
upto100nucleotides(Mockleretal.2005).Microarrayanalysiscombinedwithsuppressionsub-
tractivehybridizationhasbeenusedintheidentiicationofearlysaltstressresponsegenesintomato
root(Ouyangetal.2007;Figure12.4).
An oligonucleotide microarray is a glass chip to the surface of which an array of oligonu-
cleotides was ixed as spots, each containing numerous copies of a sequence-speciic probe that
is complementary to a gene of interest. To detect the presence of certain genes of interest in a
sample,genomicDNAisisolatedfromasample,ampliiedbyPCR,andhybridizedtothearray.
Hybridizationofthesequenceswiththeirprobesresultsonthemicroarrayandcanbedetectedbya
luorescenceimagingsystem.Theresultingpatternsandrelativeintensitiesonthemicroarraywill
showwhetherthetestedsamplesarecarryingthesecertaingenes.
Thereisatotalof20probesforGMOdetectioninaDNAmicroarraysystem,whichcanbeclas-
siiedintothreecategories.TheirstcategoryinvolvesthescreeningofGMOsfromnontransgenic
plantsbasedongeneralelementssuchaspromoter,reporter,andterminatorgenes,thesecondcat-
egoryisbasedontargetgenesequencessuchasherbicideresistance,orinsect-resistantgenes,and
thethirdcategoryscreensforspecies-speciicgenes,thatis,uniquesequencesfordifferentplant
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