1. Techniques of Molecular Biology

2. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Genomics Sequencing genomes Analyzing genome sequences Proteomics Separating proteins Analyzing proteins

3. Basic molecular biology techniques Isolating nucleic acids

4. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments

5. DNA can be reproducibly split into fragments by restriction endonucleases

6. DNA fragments can be separated by size in agarose or polyacrylamide gels Because of the phosphates in the sugar phosphate backbone, nucleic acids are negatively charged. In an electric field nucleic acids will move towards the positive pole. Smaller fragments move faster than larger fragments through the pores of a gel.

7. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments

8. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by Cloning PCR

9. DNA cloning and construction of DNA libraries Cloning in a plasmid vectorGenomic librarycDNA library

10. Vectors for DNA cloning

11. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by Cloning PCR

12. The polymerase chain reaction (PCR)

13. DNA polymerases dATP dTTP dGTP dCTP

14. DNA polymerases

15. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques

16. Single-stranded nucleic acids can bind to each other by base pairing if they contain complementary sequences Using a single-stranded labeled probe complementary base pairing is able to detect specific nucleic acids among many different nucleic acids. If the probe is used to detect DNA, the analysis is called DNA blot (Southern) analysis. If an RNA fragment is detected, the analysis is called RNA blot (northern) analysis.

17. Transcriptome analysis using microarrays

18. Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Genomics Sequencing genomes

19. Sequencing techniques dideoxysequencing pyrosequencing dATP dTTP dGTP dCTP Genomic library denature (make single-stranded) anneal primer extend primer to copy one of the strands

20. Sequencing techniques dideoxysequencing pyrosequencing

21. Sequencing techniques dideoxysequencing

22. Sequencing techniques dideoxysequencing ≈ 800 nucleotides can be sequenced in one run polyacrylamide gel electrophoresis

23. Sequencing techniques dideoxysequencing pyrosequencing ≈ 200 nucleotides can be sequenced in one run

24. Next generation sequencing methods https://en.wikipedia.org/wiki/DNA_sequencing

25. Genomics Sequencing genomes (assembling the sequence)

26. Genomics Sequencing genomes (assembling the sequence)

27. Genomics Sequencing genomes (assembling the sequence)

28. Genomics Sequencing genomes Analyzing genome sequences

29. Genomics Sequencing of genomes Split genome into pieces and sequence all pieces. Assembling the sequence (computer). Sequence analysis (annotation 1) Identify genes and other elements in sequence. Functional analysis (annotation 2) Determine function of identified elements.

30. How to find genes in a genome sequence Protein-coding genes Find open reading frames (protein-coding sequences) Find sequence with a codon bias Find upstream regulatory sequences (e.g. CpG islands) Find exon-intron boundaries Genes coding for functional RNAs Find consensus sequences for tRNAs and ribosomal RNAs Find specific RNA secondary structures (e.g. stem loops) Find upstream regulatory sequences

31. Genomic sequence

32. Finding open reading frames

33. gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtattta ctactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtga ggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccata cgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggca gaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacc tattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaaca ctatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagaca aaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaatt gtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatga acttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTA TAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctct aacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatg caaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgt ccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctatttta atactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtc agtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggc aagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaa aatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcc cgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt Expasy server Finding open reading frames

34. Sequence from the E. coli genome

35. The E. coli genome

36. 5’ UTR3’ UTR coding region = open reading frames Translation start Translation stop 5’ -- 3’ protein-coding gene = DNA transcribed into mRNA Protein-coding genes Genes = all DNA sequences that are transcribed into RNA UTR = untranslated region

37. Figure 5.4 Genomes 3 (© Garland Science 2007) Exons and introns in eukaryotic genes 5’ UTR3’ UTR

38. How to find genes in a genome sequence Protein-coding genes Find open reading frames (protein-coding sequences) Find sequence with a codon bias Find upstream regulatory sequences (e.g. CpG islands) Find exon-intron boundaries Genes coding for functional RNAs Find consensus sequences for tRNAs and ribosomal RNAs Find specific RNA secondary structures (e.g. stem loops) Find upstream regulatory sequences

39. Figure 5.6b Genomes 3 (© Garland Science 2007)

40. Figure 5.10 Genomes 3 (© Garland Science 2007) A typical sequence annotation result

41. Verifying the identity of a gene Homology search Experimental techniques Northern hybridization Zoo-blotting

42. Verifying the identity of a gene Homology search MSNQVFTTLR AATLAVILGM AGGLAVSPAQ AYPVFAQQNY ANPREANGRI VCANCHLAQK AVEIEVPQAV LPDTVFEAVI ELPYDKQVKQ VLANGKKGDL NVGMVLILPE GFELAPPDRV PAEIKEKVGN LYYQPYSPEQ KNILVVGPVP GKKYSEMVVP ILSPDPAKNK NVSYLKYPIY FGGNRGRGQV YPDGKKSNNT IYNASAAGKI VAITALSEKK GGFEVSIEKA NGEVVVDKIP AGPDLIVKEG QTVQADQPLT NNPNVGGFGQ AETEIVLQNP ARIQGLLVFF SFVLLTQVLL VLKKKQFEKV QLAEMNF BLAST BLAST = Basic Local Alignment Search Tool

43. Figure 5.28 Genomes 3 (© Garland Science 2007) 6274 ORFsCase study, yeast genome

44. Finding the function of a gene (product) Computer based analysis Homology search Experimental analysis Gene inactivation Overexpression

45. Whole genome studies Tiling assays

46. Working with proteins Separating proteins Analyzing proteins and their interactions

47. Separating proteins on polyacrylamide gels

48. Immunoblot (Western blot)

49. Proteins can be sequenced

50. Complex mixtures of proteins can be analyzed by mass spectrometry

51. Liquid chromatography is used to separate peptides before mass spectrometry

52. Mass spectrum

53. Mass spectra are compared to theoretical values

54. Figure 6.11 Genomes 3 (© Garland Science 2007) Mouse liver proteins

55. Figure 6.20a Genomes 3 (© Garland Science 2007) Protein interaction map of yeast

56. Nucleic acid protein interactions Electrophoretic mobility shift assay (EMSA)

57. Nuclease protection footprinting

58. In vitro selection assay

59. Chromatin immuno- precipitation (ChIP)

60. Chromosome conformation capture (3C assay)