Copyright IPGRI and Cornell University 2003Latest strategies 2Micr - PDF document

2 Copyright: IPGRI and Cornell University, 2003Latest strategies 2Microarray technologyDiversity array technology (DArT)Single nucleotide polymorphisms (SNPs) 3 Copyright: IPGRI and Cornell University, 2003Latest strategies 3polymorphisms within the DNA's building blocksDNA sequencin

g provides the most fundamental measure of diversity, because allimproved in recent years, and now PCR products (a DNA region amplified in sufficientquantity) can be sequenced directly and targeted to any genomic location of interest. 4 Copyright: IPGRI and Cornell University, 2003Latest

strategies 4Two main methods exist:Maxam-GilbertSanger (dideoxy sequencing or chainThe two methods differ slightly, with the Sanger method (described in slide 6) being 5 Copyright: IPGRI and Cornell University, 2003Latest strategies 5Each short piece is used as a template togenerate

a set of fragments that differ in lengthFragments are separated by gel electrophoresisidentified ('base-calling'). The original sequenceof As, Ts, Cs and Gs is recreated for each shortThe short sequences are assembled into one 6 Copyright: IPGRI and Cornell University, 2003Latest strate

gies 6 C A T A G C T G T T T C C T G T G A A AC T T TC A T A G C T G T T T C C T G T G A A AA G G A C A C T T TG C A A A G G A C A C T T TC A C T T TG T A T C G A C A A A G G A C A T T T5’3’Polymerase + dNTPs ddATP Differently coloured fluorescent dyes can be used, permitting the separ

ation of all fourfragments in a single lane on the gel and greatly increasing efficiency. Automated sequencersshows peaks representing each of the four DNA bases. 7 Copyright: IPGRI and Cornell University, 2003Latest strategies 7DNA sequencing: advantages and disadvantagesTechnically d

emandingThe results are, of course, highly reproducible and informative. Costs are high, however,many researchers. The use of PCR for targeting particular regions of DNA and the 8 Copyright: IPGRI and Cornell University, 2003Latest strategies 8Evolutionary studiesCalculations of geneti

c variationCreating PCR assays (making primers to convertAlthough marker technology is, in general, based on DNA sequence variation,fortunately, a researcher does not necessarily need to know the entire DNA sequence touse molecular markers. Of course, DNA sequencing has many useful appli

cations, but amajor drawback, particularly for diversity measurements, is that different genes evolveat different rates. Extrapolating information from particular genes to the species levelresources conservation. Pp. 85-93 Genome Mapping in Plants (H. Paterson, ed.).R.G. Landes Company,

Georgetown, TX. Maxam, A.M. and W. Gilbert. 1977. A new method for sequencing DNA. Proc. Natl Acad.Sci. U.S.A. 74:560-564.Sanger, F. 1988. Sequences, sequences and sequences. Annu. Rev. Biochem. 57:1-28.Sanger, F., S. Nicklen and A.R. Coulson. 1977. DNA sequencing with chain-terminating

inhibitors. Proc. Natl Acad. Sci. U.S.A. 74:5463-5468. 9 10 Copyright: IPGRI and Cornell University, 2003Latest strategies 10ends of an expressed gene from a cDNA libraryThis strategy is an extremely efficient way to findProgress Report, December 2001). A list of databases of ESTs for

many plants can befound at http://www.ostp.gov/NSTC/html/mpgi2001/building.htm 11 Copyright: IPGRI and Cornell University, 2003Latest strategies 11http://www.ostp.gov/NSTC/html/mpgi2001/building.htm cDNADouble-stranded DNA5’ EST3’ EST 12 Copyright: IPGRI and Cornell University, 2003Lat

est strategies 12primers for SSRsIsolation of mRNA may be difficultIntrons, which may contain importantinformation, are not part of cDNA 13 Copyright: IPGRI and Cornell University, 2003Latest strategies 13EST applications are all based on the fact thatESTs originate from segments of

gene sequences:Comparing gene diversity in different organismsGene evolution studiesSearching databases for putative orthologuesProbes for gene expression studies easier. http://www.ncbi.nlm.nih.gov/About/primer/est.html 14 15 Copyright: IPGRI and Cornell University, 2003Latest strategie

s 15Microarray, or ‘chip’, technologyMicroarrays are arrangements of small spotsof DNA fixed to glass slides or nylon membranesor hybridisation between short probes andcomplementary DNA sequencesMicroarrays are constructed using cDNAs(cDNA arrays), genomic sequences or (‘DNAGenomes can

now be analysed on a whole-genome scale, using microarray (also called'chip') technology. This technology is based on hybridisation between shortoligonucleotide probes and complementary DNA sequences. Tens of thousands ofdifferences in expression, by labelling them with different-colour

ed fluorescent dyes.Special software programs generate the data automatically. Microarrays can be used fordiagnostics, studying gene expression and gene mapping, among other things. However, 16 Copyright: IPGRI and Cornell University, 2003Latest strategies 16Center for Gene Expression

Profiling, Cornell University. http://bti.cornell.edu/CGEP/CGEP.html 17 Copyright: IPGRI and Cornell University, 2003Latest strategies 17Allow the discovery of genotype-phenotypeTechnically demandinghigh-level computing expertise and equipment 18 Copyright: IPGRI and Cornell University

, 2003Latest strategies 18Determination of expression level of genesAssay of specific genomic DNA sequenceAnalysis of expression of very largenumbers of genes (cDNA arrays)polymorphisms or SNPs) by molecularhybridisation (synthetic oligonucleotide arrays) Alscher, R. 2001. Grid it: res

ources for microarray research. http://www.bsi.vt.edu/Brown, P.O. and D. Botstein. 1999. Exploring the new world of the genome with DNARichmond, T. and S. Somerville. 2000. Chasing the dream: plant EST microarrays. 19 20 Copyright: IPGRI and Cornell University, 2003Latest strategies 20

Diversity array technology (DArT)Two steps are involved:Generating the array Genotyping a sampleDiversity arrays, also called DArT, was developed by CAMBIA. It involves a new use ofauthorisation, from CAMBIA's Web site: http://www.cambia.org.au/CAMBIA. 2000. Enabling innovation. http://w

ww.cambia.org/ 21 Copyright: IPGRI and Cornell University, 2003Latest strategies 21DArTs: preparing the array (1)the diversity of a genepool are cloned. ThePolymorphic clones in the library are identifiedby arraying inserts from a random set of clonesand hybridising the array to differ

ent samplesThe inserts from polymorphic clones are 22 Copyright: IPGRI and Cornell University, 2003Latest strategies 22DArTs: preparing the array (2) GxGyGnPool genomesthe representation Library DNAs of interest 23 Copyright: IPGRI and Cornell University, 2003Latest strategie

s 23DArTs: genotyping a sample (1)with fluorescence and hybridise against the arrayScan the array and measure, for each array spot,the amount of hybridisation signal 24 Copyright: IPGRI and Cornell University, 2003Latest strategies 24DArTs: genotyping a sample (2) GxGyChoose 2 genome

s to analyse Cut, ligate adaptorsand PCR amplify Same complexityreduction as used to makethe diversity panel Label each genomicsubset: red...Hybridise to chipLabel each genomicsubset … green 25 Copyright: IPGRI and Cornell University, 2003Latest strategies 25A hybridised DArT chip 26 C

opyright: IPGRI and Cornell University, 2003Latest strategies 26DArTs: advantages and disadvantagesFast data acquisition and analysisDetects single-base changes as well as insertions andor deletionson the enzyme used to generate the fragmentsGood transferability of markers among breedi

ngDominance of markersTechnically demanding 27 Copyright: IPGRI and Cornell University, 2003Latest strategies 27DArTs: applicationsRapid germplasm characterisationJaccoud, D., K. Peng, D. Feinstein and A. Kilian. 2001. Diversity arrays: a solid-state CAMBIA. 2000. Enabling innovation.

http://www.cambia.org/Jaccoud, D., K. Peng, D. Feinstein and A. Kilian. 2001. Diversity arrays: a solid-state 28 29 Copyright: IPGRI and Cornell University, 2003Latest strategies 29other type of polymorphismCan be identified on using microarrays andother type of marker, and are very ne

ar to or even within the gene of interest. DHPLC refers to denaturing high-performance liquid chromatography, which is used to DHPLC equipment Copyright: IPGRI and Cornell University, 2003 Latest strategies 30 31 Copyright: IPGRI and Cornell University, 2003Latest strategies 31 (top

and bottom) sharing the same sequence for 31 base pairs, except one. In position 32 Copyright: IPGRI and Cornell University, 2003Latest strategies 32Each row of yellow or blue boxes represents a single SNP. The blue boxes in each rowrepresent the major allele for that SNP, and the yel

low boxes represent the minor allele.The absence of a box at any position in a row indicates missing data.In this block, 26 common SNPs may be identified. They may be arranged in sevenpatterns include 16 of the 20 chromosomes sampled. The blue and yellow circlesPatil, N., A.J. Berno, D.A

. Hinds, W.A. Barret, J.M. Doshi, C.R. Hacker, and others. 2001.chromosome 21. Science 294:1719-1723.., 2001. Science 294:1719-1723.SNP #2 33 Copyright: IPGRI and Cornell University, 2003Latest strategies 33Strategies are continuously being developed to improvethe detection of polymor

phismsDNA sequencing allows the detection of variation at eventhe nucleotide levelESTs are powerful tools for detecting diversity withinMicroarrays and DArT make the simultaneous analysis of 34 Copyright: IPGRI and Cornell University, 2003Latest strategies 34By now you should know The

different types of DNA variation that can be The underlying principle of microarrays and DArT The advantages and disadvantages of the newesttechnologies for analysing genetic diversity Griffiths, A.J.F., J.H. Miller, D.T. Suzuki, R.C. Lewontin and W.M. Gelbart. 1996. Anintroduction to ge

netic analysis (6th edn.). W.H. Freeman and Co., NY.Hajeer, A., J. Worthington and S. John (eds.). 2000. SNP and Microsatellite Genotyping:Eaton Publishing, Manchester, UK.USDA-ARS. 1999. The Cregan lab. http://bldg6.arsusda.gov/pberkum/Public/sarl/cregan/Wang, D.G., J.B. Fan, C.J. Siao,

A. Berno, P. Young, R. Sapolsky, and others. 1998. 35 36 Copyright: IPGRI and Cornell University, 2003Latest strategies 36Glossary Using molecular marker technology instudies on plant genetic diversity(DNA sequencing, ESTs, microarrays, DArT, SNPs) Copyright: IPGRI and Cornell Univers