Combining SELEX with Combining SELEX with quantitative assays to rapidly quantitative assays to rapidly

obtain accurate models of obtain accurate models of protein–DNA interactionsprotein–DNA interactions

Jiajian Liu and Gary D. StormoJiajian Liu and Gary D. Stormo

Presented by Aliya SadequePresented by Aliya Sadeque

Protein-DNA interactionsProtein-DNA interactions

Methods for measuring:Methods for measuring:Yeast 1 hybridYeast 1 hybridChIP on chipChIP on chipDNA microarrayDNA microarray

Important distinction in terms of specificityImportant distinction in terms of specificityEnzymes vs. transcription factorsEnzymes vs. transcription factors

Bioinformatics. 2000 Jan;16(1):16-23.

TFBSTFBSTranscription Factor Binding SitesTranscription Factor Binding Sites

Knowing the specificity of a TF in order to Knowing the specificity of a TF in order to locate its binding sites within the genome locate its binding sites within the genome

DatabasesDatabasesTRANSFAC, JASPARTRANSFAC, JASPAR

Represented as Represented as consensus sequencesconsensus sequences or or weight matricesweight matrices

SELEXSELEXSystematic Evolution of Ligands By Exponential EnrichmentSystematic Evolution of Ligands By Exponential Enrichment

http://rulai.cshl.edu/tools/ESE2/ESEmatrix.html

QuMFRAQuMFRA

Nucleic Acids Res. 2001 Jun 15;29(12):2471-8. PMID: 11410653

Selling pointsSelling points

Provides a general Provides a general method that can be method that can be used for any DNA-used for any DNA-binding protein even if binding protein even if nothing is known nothing is known about its specificity. about its specificity.

Can isolate a small Can isolate a small set of specific binding set of specific binding sites from a very large sites from a very large pool of random pool of random sequencessequences

Nucleic Acids Res. 2001 Jun 15;29(12):2471-8.

Zif268Zif268

SELEX ProcedureSELEX Procedure

Estimating AffinitiesEstimating Affinities

Assuming an additive modelAssuming an additive model Frequency:Frequency:

WeightWeight

Sequence LogoSequence Logo

QuMFRA ProcedureQuMFRA Procedure

15 sequences15 sequencesCover the space of possible sequenceCover the space of possible sequence

http://www.answers.com/topic/gel-abpp-eg-png

Uh oh, math.Uh oh, math.

critiquescritiques

we used affinity chromatography to modify we used affinity chromatography to modify the conventional SELEX procedure to the conventional SELEX procedure to save time and labor. –didn’t explain howsave time and labor. –didn’t explain how

as determined by sequencing only 20 ∼as determined by sequencing only 20 ∼selected products after each round. –why selected products after each round. –why only 20?only 20?

Why no A at position 1 in QuMFRA?Why no A at position 1 in QuMFRA?Discussion brought up weakness in Discussion brought up weakness in

SELEXSELEX