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Jiajian Liu and Gary D. Stormo Presented by Aliya Sadeque

Combining SELEX with quantitative assays to rapidly obtain accurate models of protein–DNA interactions. Jiajian Liu and Gary D. Stormo Presented by Aliya Sadeque. Protein-DNA interactions. Methods for measuring: Yeast 1 hybrid ChIP on chip, DNA microarray

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Jiajian Liu and Gary D. Stormo Presented by Aliya Sadeque

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  1. Combining SELEX with quantitative assays to rapidly obtain accurate models of protein–DNA interactions Jiajian Liu and Gary D. Stormo Presented by Aliya Sadeque

  2. Protein-DNA interactions • Methods for measuring: • Yeast 1 hybrid • ChIP on chip, DNA microarray • Important distinction in terms of specificity • Enzymes vs. transcription factors Bioinformatics. 2000 Jan;16(1):16-23.

  3. TFBSTranscription Factor Binding Sites • Goal: Knowing the specificity of a TF in order to locate its binding sites within the genome • Sites represented as consensus sequences or weight matrices • Databases: TRANSFAC, JASPAR

  4. SELEXSystematic Evolution of Ligands By Exponential Enrichment http://rulai.cshl.edu/tools/ESE2/ESEmatrix.html

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

  6. Selling points • Provides a general method that can be used for any DNA-binding protein even if nothing is known about its specificity. • Can isolate a small set of specific binding sites from a very large pool of random sequences Nucleic Acids Res. 2001 Jun 15;29(12):2471-8.

  7. Zif268

  8. SELEX Procedure

  9. SELEX Binding Model • Assuming an additive model • Frequency: • Weight

  10. Sequence Logoobtained from SELEX

  11. QuMFRA Procedure • 15 sequences • Cover the space of possible sequence • Competitive binding assay http://www.answers.com/topic/gel-abpp-eg-png

  12. Uh oh, math. • Intensities of each DNA in a separated band • Obtained from emission matrix and output vector • Relative binding constant of a test site with respect to a reference site • Reference was GGGT bound unbound

  13. QuMFRA Binding Model Weight matrix Consensus Sequence Matrix values are proportional to binding affinity according to the Berg and von Hippel theory.

  14. Comparing notesBetween SELEX and QuMFRA • Experimental Kafor 15 sequences • Renormalizedon consensussequence

  15. Comparing notesBetween predictions and empirical binding affinities • Found affinity measures for 8 variants of the consensus sequence with one or two changed positions

  16. A Probabilitistic Recognition CodeI promise those are all real words

  17. AlternativesSAGE-SELEX • Improves on the number of binding sites found by SELEX alone • Large sample size reqired for statistical significance • Biases in SELEX

  18. Alternatives dsDNA chips • Chips contain binding sites for a TF of interest • High throughput quantitative data • Almost all possible binding sites would have to be on the chip…that’s a lot.

  19. AlternativesSimilar Sequences • optimized selection of DNA variants to be tested experimentally • quantitative protein-DNA binding assay • prediction of binding affinity for all variants using a statistical model • Can be done in high throughput with high accuracy, provided the consensus sequence is known

  20. Praises General: • Can be used for a TF of unknown specificity and size • Efficient • Parallel (different colours of fluorophore) • First step narrows down sample size

  21. Heckles • Sequenced a sample of ~20 - too small. • Could skew data. Secondary preference? • Why no A at position 1 in QuMFRA? • Weakness/inherent bias in SELEX • Empirical data - is an average appropriate?

  22. Heckles • Correlation values (0.54, 0.6, 0.77, 0.94) – are these significant? No T-test or anything?

  23. Questions?

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