Basic Molecular Biology

1. Basic Molecular Biology Many slides by Omkar Deshpande

2. Overview • Structures of biomolecules • Central Dogma of Molecular Biology • Overview of this course • Genome Sequencing

3. Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001

5. Watson and Crick

8. Nucleic acids (DNA and RNA) • Form the genetic material of all living organisms. • Found mainly in the nucleus of a cell (hence “nucleic”) • Contain phosphoric acid as a component (hence “acid”) • They are made up of nucleotides.

9. Nitrogenous Base Nitrogenous Base Phosphate Group Phosphate Group Sugar Sugar Nucleotides

10. A T G C C G G C A T C G A T G C DNA A = T G = C

11. The gene and the genome • Genome = The entire DNA sequence within the nucleus. • The information in the genome is used for protein synthesis • A gene is a length of DNA that codes for a (single) protein.

12. How big are genomes?

13. Repeats • The DNA is full of repetitive elements (those that occur over & over & over) • There are several type of repeats, including SINEs & LINEs (Short & Long Interspersed Elements) (1 million just ALUs) and low complexity elements. • Their function is poorly understood, but they make problems more difficult.

14. Central dogma ZOOM IN tRNA transcription DNA rRNA snRNA translation POLYPEPTIDE mRNA

15. Transcription • The DNA is contained in the nucleus of the cell. • A stretch of it unwinds there, and its message (or sequence) is copied onto a molecule of mRNA. • The mRNA then exits from the cell nucleus.

16. A A T G G C C C G G G C A A T C C G U A T G G C DNA RNA A = T G = C T  U

17. More complexity • The RNA message is sometimes “edited”. • Exons are nucleotide segments whose codons will be expressed. • Introns are intervening segments (genetic gibberish) that are snipped out. • Exons are splicedtogether to form mRNA.

18. Splicing frgjjthissentencehjfmkcontainsjunkelm thissentencecontainsjunk

19. Key player: RNA polymerase • It is the enzyme that brings about transcription by going down the line, pairing mRNA nucleotides with their DNA counterparts.

20. Promoters • Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation. • The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated. 5’ 3’ Promoter

21. Promoters • Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation. • The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated. 5’ 3’ Promoter

22. Transcription – key steps DNA • Initiation • Elongation • Termination DNA + RNA

23. Transcription – key steps DNA • Initiation • Elongation • Termination

24. Transcription – key steps DNA • Initiation • Elongation • Termination

25. Transcription – key steps DNA • Initiation • Elongation • Termination

26. Transcription – key steps DNA • Initiation • Elongation • Termination DNA + RNA

27. Genes can be switched on/off • In an adult multicellular organism, there is a wide variety of cell types seen in the adult. eg, muscle, nerve and blood cells. • The different cell types contain the same DNA though. • This differentiation arises because different cell types express different genes. • Promoters are one type of gene regulators

28. Transcription (recap) • The DNA is contained in the nucleus of the cell. • A stretch of it unwinds there, and its message (or sequence) is copied onto a molecule of mRNA. • The mRNA then exits from the cell nucleus. • Its destination is a molecular workbench in the cytoplasm, a structure called a ribosome.

29. Translation • How do I interpret the information carried by mRNA to the Ribosome? • Think of the sequence as a sequence of “triplets”. • Think of AUGCCGGGAGUAUAG as AUG-CCG-GGA-GUA-UAG. • Each triplet (codon) maps to an amino acid.

30. The Genetic Code • f : codon amino acid • 1968 Nobel Prize in medicine – Nirenberg and Khorana • Important – The genetic code is universal! • It is also redundant / degenerate.

31. The Genetic Code

32. Proteins • Composed of a chain of amino acids. R | H2N--C--COOH | H 20 possible groups

33. Proteins R R | | H2N--C--COOH H2N--C--COOH | | H H

34. Dipeptide This is a peptide bond R O R | II | H2N--C--C--NH--C--COOH | | H H

35. Protein structure • Linear sequence of amino acids folds to form a complex 3-D structure. • The structure of a protein is intimately connected to its function. The 3-D shape of proteins gives them their working ability – the ability to bind with other molecules.

36. Our course (2417) Part 1, DNA: Assembly, Evolution, Alignment Part 2, Genes: Prediction, Regulation transcription DNA rRNA snRNA translation POLYPEPTIDE mRNA

37. DNA Sequencing Some slides shamelessly stolen from Serafim Batzoglou

38. DNA sequencing How we obtain the sequence of nucleotides of a species …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

39. Which representative of the species? Which human? Answer one: Answer two: it doesn’t matter Polymorphism rate: number of letter changes between two different members of a species Humans: ~1/1,000 – 1/10,000 Other organisms have much higher polymorphism rates

40. Why humans are so similar A small population that interbred reduced the genetic variation Out of Africa ~ 40,000 years ago Out of Africa

41. Migration of human variation http://info.med.yale.edu/genetics/kkidd/point.html

42. http://info.med.yale.edu/genetics/kkidd/point.html Migration of human variation

43. http://info.med.yale.edu/genetics/kkidd/point.html Migration of human variation

44. DNA Sequencing Goal: Find the complete sequence of A, C, G, T’s in DNA Challenge: There is no machine that takes long DNA as an input, and gives the complete sequence as output Can only sequence ~500 letters at a time

45. DNA sequencing – vectors DNA Shake DNA fragments Known location (restriction site) Vector Circular genome (bacterium, plasmid) + =

46. DNA sequencing – gel electrophoresis • Start at primer (restriction site) • Grow DNA chain • Include dideoxynucleoside (modified a, c, g, t) • Stops reaction at all possible points • Separate products with length, using gel electrophoresis

47. Electrophoresis diagrams

48. Challenging to read answer

49. Challenging to read answer

50. Reading an electropherogram • Filtering • Smoothening • Correction for length compressions • A method for calling the letters – PHRED PHRED – PHil’s Read EDitor (by Phil Green) Based on dynamic programming Several better methods exist, but labs are reluctant to change