Chemistry Reference
In-Depth Information
Restriction enzymes
E
E
M
E
E
M
E
M
M
M
Eco
RI
+Mse
I
M
E
Genomic DNA
M
M
Adapter
ligation
E
M
M
M
E:
EcoRI restriction site
M:
MseI restriction site
End-specific; DNA
adapters
M
M
E
M
:
M
M
Figure 12.2
AFLP analysis employs the digestion of genomic DNA with
Mse
I and
Eco
RI enzymes. The
resulting fragments are ligated to end-speciic adapter molecules and are used in a preselective
PCR with primers complementary to each of the two adapter sequences having an additional
base at the 3
ʹ
end. Ampliication of only 1/16 of
Eco
RI-
Mse
I fragments occurs. (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.)
12.4.1.2 Ampliied Fragment Length Polymorphism
Theampliiedfragmentlengthpolymorphism(AFLP)techniqueisbasedontheselectivePCR
ampliicationofrestrictionfragmentsfromatotaldigestofgenomicDNA.Thetechniqueinvolves
threesteps:(1)restrictionoftheDNAandligationofoligonucleotideadapters;(2)selectiveampli-
icationofsetsofrestrictionfragments;and(3)gelanalysisoftheampliiedfragments.PCRampli-
ication of restriction fragments is achieved by using the adapter and restriction site sequence as
targetsitesforprimerannealing.Theselectiveampliicationisachievedbytheuseofprimersthat
extendintotherestrictionfragments,amplifyingonlythosefragmentsinwhichtheprimerexten-
sionsmatchthenucleotideslankingtherestrictionsites.Withthismethod,setsofrestrictionfrag-
mentsaredeterminedwithoutpriorknowledgeofthenucleotidesequence.Themethodallowsthe
speciiccoampliicationofhighnumbersofrestrictionfragmentsandisusuallyaccompaniedwith
automated capillary electrophoresis (Vos et al. 1995). AFLP, simple sequence repeat, and single
nucleotidepolymorphismhavebeenappliedtothetomatogenomefortheassessmentofpolymor-
phismandforgenomemapping(Suliman-Pollatscheketal.2002),whilenewAFLPsequencesare
constantlyaddedattheGenBankdatabase(Argyropoulosetal.2008;Figure12.2).
12.4.1.3 Serial Analysis of Gene Expression
Serial analysis of gene expression (SAGE) was developed as an elegant means of analyzing
mRNApopulationsbythelarge-scalesequencedeterminationofshortidentifyingstretchesofindi-
vidualmessengers(Velculescuetal.1995);however,therequireddepthofsequencingunderdiffer-
entconditionsmakesitalsolaborintensiveandtimeconsuming(Figure12.3).
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