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.
Very large DNA molecules are separated from each other by special types of electrophoresis, e.g. pulsed field electrophoresis.

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 vector
Genomic library
cDNA 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. DNA polymerases
dATP
dTTP
dGTP
dCTP

13. DNA polymerases

14. The polymerase chain reaction (PCR)

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
26x24 = 624 spots

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
2’ deoxynucleotide
2’-3’-dideoxynucleotide
Base
Base

21. Sequencing techniques
dideoxysequencing
2’ deoxynucleotide
2’-3’-dideoxynucleotide
Base
Base

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

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

24. Next generation sequencing methods

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. Finding open reading frames
gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtatttactactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtgaggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccatacgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggcagaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacctattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaacactatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagacaaaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaattgtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatgaacttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTATAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctctaacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatgcaaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctattttaatactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtcagtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggcaagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaaaatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcccgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt
Expasy server

34. Sequence from the E. coli genome

35. The E. coli genome
High gene density on both strands of the E. coli genome.

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

37. Exons and introns in eukaryotic genes
5’ UTR
3’ UTR
Features that can be used to find genes in eukaryotic sequences:
Codon bias.
Exon-intron boundaries.
Upstream sequences: in vertebrates CpG islands (in 40-50% of human genes).
Figure 5.4 Genomes 3 (© Garland Science 2007)

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. A typical sequence annotation result
Automatic annotation, 15 kb sequence of the human genome containing a tissue factor gene. Location of 6 exons revealed.
Figure Genomes 3 (© Garland Science 2007)

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

42. Verifying the identity of a gene
Homology search
BLAST
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 = Basic Local Alignment Search Tool

43. Case study, yeast genome 6274 ORFs
Orphans= genes of unknown function; single orphans = unique genes not found in databases.
Additional methods for identifying the function of genes:
Comparative genomics.
cDNA sequencing.
Transposon tagging.
Figure Genomes 3 (© Garland Science 2007)

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. Proteomics Isolating and separating proteins
Identifying and analyzing proteins

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

48. Separating proteins on polyacrylamide gels

49. Immunoblot (Western blot)

50. Proteins can be sequenced

51. Complex mixtures of proteins can be analyzed by mass spectrometry
MALDI-TOF = Matrix-assisted laser desorption ionization – time of flight

52. Typical workflow in analysis of proteins by mass spectometry

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

54. Mass spectrum

55. Mass spectra are compared to theoretical values

56. Mouse liver proteins
Example of spots on a gel obtained after 2D electrophoresis.
Spots of interest are cut out and protein identified by mass spectrometry.
Figure Genomes 3 (© Garland Science 2007)

57. Protein interaction map of yeast
Each dot is a protein. Red dot: essential protein.
Figure 6.20a Genomes 3 (© Garland Science 2007)

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

59. Nuclease protection footprinting is used to identify the DNA sequence to which a protein binds

60. In vitro selection assay uses a combinatorial DNA sequence library to identify DNA sequences to which a protein binds.

61. Chromatin immuno-
precipitation (ChIP)
Identifies protein-binding sites in vivo

62. Chromosome
conformation
capture (3C assay)
Identifies DNA sequences that are conformationally linked