BME280/CSE277/CSE377: Bioinformatics Spring 2006

1. BME280/CSE277/CSE377: BioinformaticsSpring 2006

2. Administrivia • Lecture time: TTh 12:30-1:45pm • Lecture place: Engineering II, Room 322 • Instructor: Ion Mandoiu • Office: ITEB 261 • Tel: 6-3784 • E-mail: ion@engr.uconn.edu • Office hours: MW 1-2pm

3. Textbooks • Neil C. Jones and Pavel A. Pevzner, An Introduction to Bioinformatics Algorithms, MIT Press, 2004. Textbook website: http://bioalgorithms.info/. (REQUIRED) • D. Gusfield, Algorithms on Strings, Trees, and Sequences, Cambridge University Press, 1997 (OPTIONAL)

4. Grading • 30% homework assignments • Bi-weekly • 30% programming projects • Individual, 3-4 projects • 40% final project • Individual or teams of 2 • Written report + short presentation • Possible topics • Algorithm implementation + empirical study • In-depth survey of a topic not covered in class • Progress on open research problems • Propose your own!

5. What is Bioinformatics? • Bioinformatics is generally defined as the analysis, prediction, and modeling of biological data with the help of computers

6. Why Bioinformatics? • DNA sequencing technologies have created massive amounts of information that can only be efficiently analyzed with computers • Hundreds of species sequenced • Human, rat, chimp, chicken, … • As the information becomes ever so larger and more complex, more computational tools are needed to sort through the data. • Biology is becoming an information science! • Slowly, we are learning how cells work through comparative genomics -- not unlike comparative linguistics

7. Bioinformatics Tools • Bioinformatics problems involve multiple aspects • Example: Sequence Comparison • Biology: How are genes evolving? How is gene function related to gene sequence? • Learning/AI: How do we define “similar’’? Can we learn from examples? • Algorithms: How can we efficiently find all similar sequences? • Statistics: How do we distinguish a random match from a true one?

8. Course Description • Course emphasis • Modeling computational problems arising in biology as graph-theoretic, statistical, or mathematical optimization problems • Design, analysis, and implementation of efficient algorithms • Algorithmic techniques to be covered • Exhaustive search • Integer programming • Greedy algorithms • Dynamic programming • Divide-and-conquer • Graph algorithms • Combinatorial pattern matching • Clustering • Hidden Markov models • Randomized algorithms

9. Course Description • Biological applications • Restriction mapping • DNA sequencing • Motif finding • Pairwise sequence alignment • Gene prediction • Evolutionary trees • Genome rearrangements

10. Complete and return the survey!

11. Basic Molecular Biology

12. The Cell Source: D. Geiger All cells contain the same DNA, yet there are many types of cells!

13. Mendel and his Genes • Genes -- physical and functional traits passed on from one generation to the next • Discovered by Gregor Mendel in the 1860s while he was experimenting with the pea plant. He asked the question: Do traits come from a blend of both parent's traits or from only one parent?

14. The Pea Plant Experiments • Mendel discovered that genes were passed on to offspring by both parents in two forms: dominant and recessive. • The dominant form would be the phenotypic characteristic of the offspring

15. DNA: The Code of Life • The structure and the four genomic letters code for all living organisms • Adenine, Guanine, Thymine, and Cytosine which pair A-T and C-G on complimentary strands.

16. DNA Components Source: D. Geiger

17. The Human Genome Source: D. Geiger

18. DNA Organization Source: D. Geiger

19. Genome Sizes • E. Coli (bacteria) 4.7 Mb (Mega bases) • Yeast (simple fungi) 15 Mb • Nematode (C. Elegans) 100 Mb • Mouse 2 Gb (Giga bases) • Human 3 Gb • Wheat 16.5 Gb • Lily 32-48 Gb

20. Genes • DNA strings contain: • Coding regions (genes) • Control regions • “Junk” DNA (unknown function) • Estimated number of genes: • E. Coli (bacteria) 4,000 • Yeast (simple fungi) 6,000 • Nematode (C. Elegans) 13,000 • Human 32,000 (?)

21. Central Dogma • Cells express different subsets of genes under different environments Translation Transcription Protein mRNA Gene

22. Gene Transcription Source: D. Geiger • RNA: similar to DNA, but has • slightly different backbone • Uracil (U) instead of Thymine (T)

23. RNA Roles Source: D. Geiger

24. Translation • Catalyzed by Ribosome • Using two different sites, the Ribosome continually binds tRNA, joins the amino acids together and moves to the next location along the mRNA • ~10 codons/second, but multiple translations can occur simultaneously http://wong.scripps.edu/PIX/ribosome.jpg

25. Genetic Code • Human cells produce approx. 100,000 proteins • Proteins are poly-peptides consisting of 70-3,000 amino acids • There are 20 different amino acids; every 3 nucleotides in a gene encode for 1 amino acid (or the STOP signal) Source: D. Geiger

26. Protein Folding • Proteins are not linear structures, though they are built that way • The amino acids have very different chemical properties; they interact with each other after the protein is built • This causes the protein to start fold and adopting it’s functional structure • Proteins may fold in reaction to some ions, and several separate chains of peptides may join together through their hydrophobic and hydrophilic amino acids to form a polymer

27. Protein Folding (cont’d) • The structure that a protein adopts is vital to it’s chemistry • Its structure determines which of its amino acids are exposed carry out the protein’s function • Its structure also determines what substrates it can react with

28. Protein Structure Source: D. Geiger

29. Basic Molecular BiotechnologyHow is information accessed at molecular level?

30. Operations on DNA/RNA • Amplification (making many copies) • Cutting into shorter fragments • Reading fragment lengths • Reading DNA sequence • Probing presence of specific fragments

31. Why we need so many copies • Biologists needed to find a way to read DNA codes. • How do you read base pairs that are angstroms in size? • It is not possible to directly look at it due to DNA’s small size. • Need to use chemical techniques to detect what you are looking for. • To read something so small, you need a lot of it, so that you can actually detect the chemistry. • Need a way to make many copies of the base pairs, and a method for reading the pairs.

32. Polymerase Chain Reaction • Problem: Modern instrumentation cannot easily detect single molecules of DNA, making amplification a prerequisite for further analysis • Solution: PCR doubles the number of DNA fragments at every iteration 1… 2… 4… 8…

33. Denaturation Raise temperature to 94oC to separate the duplex form of DNA into single strands

34. Design primers • To perform PCR, a 10-20bp sequence on either side of the sequence to be amplified must be known because DNA pol requires a primer to synthesize a new strand of DNA

35. Annealing • Anneal primers at 50-65oC

36. Annealing • Anneal primers at 50-65oC

37. Extension • Extend primers: raise temp to 72oC, allowing Taq pol to attach at each priming site and extend a new DNA strand

38. Extension • Extend primers: raise temp to 72oC, allowing Taq pol to attach at each priming site and extend a new DNA strand

39. Repeat • Repeat the Denature, Anneal, Extension steps at their respective temperatures…

40. Polymerase Chain Reaction

41. Restriction Enzymes • Discovered in the early 1970’s • Used as a defense mechanism by bacteria to break down the DNA of attacking viruses. • They cut the DNA into small fragments. • Can also be used to cut the DNA of organisms. • This allows the DNA sequence to be in a more manageable bite-size pieces. • It is then possible using standard purification techniques to single out certain fragments and duplicate them to macroscopic quantities.

42. Molecular Scissors Molecular Cell Biology, 4th editionfig 9-10

43. Discovering Restriction Enzymes • HindII: first restriction enzyme discovered by Hamilton Smith in 1970 • From bacterium Haemophilus influenzae • Discovered accidentally while studying how the bacterium Haemophilus influenzae takes up DNA from the phage virus P22 • Recognizes and cuts DNA at sequences: • GTGCAC • GTTAAC

44. Recognition Sites of Restriction Enzymes

45. Separating DNA by Size • Gel electrophoresis is a process for separating DNA by size • Can separate DNA fragments that differ in length in only 1 nucleotide for fragments up to 500 nucleotides long

46. Gel Electrophoresis • DNA fragments are injected into a gel positioned in an electric field • DNA are negatively charged near neutral pH • The ribose phosphate backbone of each nucleotide is acidic; DNA has an overall negative charge • DNA molecules move towards the positive electrode

47. Gel Electrophoresis (cont’d) • DNA fragments of different lengths are separated according to size • Smaller molecules move through the gel matrix more readily than larger molecules • The gel matrix restricts random diffusion so molecules of different lengths separate into bands

48. Detecting DNA: Autoradiography • One way to visualize separated DNA bands on a gel is autoradiography: • The DNA is radioactively labeled • The gel is laid against a sheet of photographic film in the dark, exposing the film at the positions where the DNA is present.

49. Detecting DNA: Fluorescence • Another way to visualize DNA bands in gel is fluorescence: • The gel is incubated with a solution containing the fluorescent dye ethidium • Ethidium binds to the DNA • The DNA lights up when the gel is exposed to ultraviolet light.

50. Gel Electrophoresis: Example Direction of DNA movement Smaller fragments travel farther