1. Molecular Diagnostics 1 1 Analysis and Characterization of Nucleic Acids and Proteins

2. Molecular Diagnostics2 RESTRICTION NUCLEASE Different species of bacteria make different restriction nucleases, which protect them from viruses by degrading incoming viral DNA. Each nuclease recognizes a specific sequence of four to eight nucleotides in DNA. These sequences, where they occur in the genome of the bacterium itself, are protected from cleavage by methylation at an A or a C residue

3. Molecular Diagnostics3 Type 1 restriction enzymes Have both nuclease and methylase activity Complex enzymes with two subunits. They bind to sites of 4–6 bp separated by 6–8 bp and containing methylated adenines. The site of cleavage can be over 1000 bp from this binding site. An example is EcoK from E. coli K 12.  recognizes the site: 5 ‘- A C N N N N N N G T G C - 3’ 3’ - T G N N N N N N C A C G – 5’  and adenine residues (A) are methylated Molecular Diagnostics 3

4. 4 Type II restriction enzymes Used most frequently in the laboratory. Do not have methylation activity Bind as simple dimers to their symmetrical DNA recognition sites (palindromic or bilateral symmetry) Cleave the DNA directly at their binding site, producing fragments of predictable size. Molecular Diagnostics 4

5. 5 Type III restriction enzymes Able to both methylate and restrict (cut) DNA. Complex enzymes with two subunits. Recognition sites are asymmetrical Cleavage occurs 24–26 bp from recognition site to the 3 side. Example is HinfIII from H. influenzae.  It recognizes the site: 5’- C G A A T – 3’ 3’- G C T T A – 5’  adenine methylation occurs on only one strand. Molecular Diagnostics 5

6. 6 6

7. 7 7 BamH1 GGATCC CCTAGG HaeIII GGCC CCGG Cohesive Ends (5 ´ Overhang) Cohesive Ends (3 ´ Overhang) KpnI GGTACC CCATGG Blunt Ends (No Overhang) Restriction Endonucleases: Type II

8. Molecular Diagnostics8 8 GATC CTAG DpnI (Requires methylation) Methylation-sensitive Enzymes GGCC CCGG HaeIII (Inhibited by methylation) CCCGGG GGGCCC XmaI (5’ Overhang) CCCGGG GGGCCC SmaI (Blunt Ends) Isoschizomers Enzymes Generating Compatible Cohesive Ends GGATCC CCTAGG BamHI (5’ Overhang) AGATCT TCTAGA BglII (5’ Overhang) CTCGTG GAGCAG BssSI (5’ Overhang) NNCAGTGNN NNGTCACNN TspRI (3’ Overhang) Enzymes Recognizing Non palindromic Sequences Restriction Endonucleases: Type II

9. Molecular Diagnostics9 DNA ligase Catalyzes the formation of a phosphodiester bond between adjacent 3-hydroxyl and 5- phosphoryl nucleotide ends. Molecular Diagnostics 9

10. 10 GAATTC caccgtgGAATTCacgaacaa CTTAAG gtggcacCTTAAGtgcttgtt GAATTC acaacgaGAATTCctttatc CTTAAG tgttgctCTTAAGgaaatag EcoRI caccgtgGAATTCctttatc gtggcacCTTAAGgaaatag ligase + ATP caccgtgG AATTCacgaacaa gtggcacCTTAA Gtgcttgtt P OH HO P ligase + ATP caccgtgGAATTCtcgttgt gtggcacCTTAAGagcaaca acaacgaG AATTCctttatc tgttgctCTTAA Ggaaatag P OH HO P Restriction fragments can be religated ligase + ATP

11. Molecular Diagnostics11 caccgtgG AATTCacgaacaa gtggcacCTTAA Gtgcttgtt P OH HO P acaacgaG AATTCctttatc tgttgctCTTAA Ggaaatag P OH HO P caccgtgG AATTCacgaacaa gtggcacCTTAA Gtgcttgtt OH HO caccgtgG AATTCacgaacaa gtggcacCTTAA Gtgcttgttphosphatase Cannot be ligated Nick Cannot be ligated, but can be replicated caccgtgGAATTCctttatc gtggcacCTTAAGgaaatag Self ligation can be prevented ligase + ATP

12. Molecular Diagnostics12 GAATTC caccgtgG AATTCacgaacaa CTTAAG gtggcacCTTAA Gtgcttgtt DNA pol + dNTPs GAATTAATTC caccgtgGAATTAATTCacgaacaa CTTAATTAAG gtggcacCTTAATTAAGtgcttgtt GAATTAATTC caccgtgGAATT AATTCacgaacaa CTTAA TTAAG gtggcacCTTAA TTAAGtgcttgtt ligase + ATP The ends can be modified

13. Molecular Diagnostics13 Some enzymes have different recognition sites, but create compatible cohesive ends caccgtgGGATCCacgaacaa gtggcacCCTAGGtgcttgtt acaacgaAGATCTctttatc tgttgctTCTAGAgaaatag acaacgaA GATCTctttatc tgttgctTCTAG Agaaatag P OH HO P Bgl II caccgtgG GATCCacgaacaa gtggcacCCTAG Gtgcttgtt P OH HO P BamHI caccgtgGGATCTctttatc gtggcacCCTAGAgaaatag ligase + ATP BamHI BamHI does not cut BglII BglII does not cut compatible cohesive ends

14. Molecular Diagnostics14 Molecular Diagnostics 14 Restriction Enzyme Mapping Digest DNA with a restriction enzyme. Resolve the fragments by gel electrophoresis. The number of bands indicates the number of restriction sites. The size of the bands indicates the distance between restriction sites.

15. Molecular Diagnostics15 Restriction Enzyme Mapping Molecular Diagnostics 15 Two possible maps inferred from the observations

16. Molecular Diagnostics16 Molecular Diagnostics 16 Restriction Enzyme Mapping 4.3 kb 3.7 kb 2.3 kb 1.9 kb 1.4 kb 1.3 kb 0.7 kb BamH1 XhoI BamH1 XhoI

17. Molecular Diagnostics17 Homework? HindIIISal I Determine the relative positions of HindIII and Sal I on this piece of DNA

18. Molecular Diagnostics18 Molecular Diagnostics 18 Hybridization Technologies

19. Molecular Diagnostics19 Molecular Diagnostics 19 Blots Southern blots  DNA immobilized on solid support Northern blots  RNA immobilized on solid support Western blots  Proteins immobilized on solid support

20. Molecular Diagnostics20

21. Molecular Diagnostics21 Molecular Diagnostics 21 Southern Blot Hybridization Developed by Edwin Southern. The Southern blot procedure allows analysis of any specific gene or region without having to clone it from a complex background.  Transfer DNA from a gel matrix to a filter (nitrocellulose, nylon)  Fix DNA to filter (Heat under a vacuum, UV cross-link  Block with excess DNA (unrelated)  Hybridize with labeled DNA probe  Wash unbound probe (controls stringency)

22. Molecular Diagnostics22 Blotting a Gel

23. Molecular Diagnostics23 DNA Binding Solid Support Electrostatic and hydrophobic:  Nitrocellulose  Nylon  Reinforced nitrocellulose Electrostatic  Nylon, nytran  Positively charged nylon Molecular Diagnostics 23

24. 24 Molecular Diagnostics 24 Transfer of DNA to Membrane: Capillary Transfer

25. Molecular Diagnostics25 Molecular Diagnostics 25 Transfer of DNA to Membrane: Electrophoretic Transfer

26. Molecular Diagnostics26 Molecular Diagnostics 26 Transfer of DNA to Membrane: Vacuum Transfer

27. Molecular Diagnostics27 Nucleic Acid Hybridization Takes advantage of the ability of individual single- stranded nucleic acid molecules to form double- stranded molecules (that is, to hybridize to each other). Can occur between any two single-stranded nucleic acid chains (DNA/DNA, RNA/RNA, or RNA/DNA). The interacting single-stranded molecules must have a sufficiently high degree of base complementarity.

28. Molecular Diagnostics28 Molecular Diagnostics 28 Denaturation/Annealing: An Equilibrium Reaction

29. Molecular Diagnostics29 Molecular Diagnostics 29 Denaturation and Annealing of DNA

30. Molecular Diagnostics30 Melting Temperature (T m ) The temperature at which 50% of a nucleic acid is hybridized to its complementary strand. Molecular Diagnostics 30

31. 31 Melting Temperature and Hybridization Your hybridization results are directly related to the number of degrees below the melting temperature (T m ) of DNA at which the experiment is performed. For an aqueous solution of DNA (no salt) the formula for Tm is: Tm = 69.3 o C + 0.41(% GC) o C Molecular Diagnostics 31

32. 32 Factors affecting melting temperature The energy required to separate two perfectly complementary DNA strands is dependent on a number of factors, notably:  Strand length - long homoduplexes contain a large number of hydrogen bonds and require more energy to separate them;  Base composition – sequences with high % GC composition are more difficult to separate than those with a low % GC composition;  Chemical environment - the presence of monovalent cations (e.g. Na+ ions) stabilizes the duplex, whereas chemical denaturants (such as formamide and urea) destabilize the duplex by chemically disrupting the hydrogen bonds.

33. Molecular Diagnostics33 Molecular Diagnostics 33 Melting Temperatures: base composition Sequences with high % GC composition are more difficult to separate than those with a low % GC composition The following examples, demonstrate the point.  Tm = 69.3 o C + 0.41(45) o C = 87.5 o C  Tm = 69.3 o C + 0.41(40) o C = 85.7 o C  Tm = 69.3 o C + 0.41(60) o C = 93.9 o C

34. Molecular Diagnostics34 Melting Temperatures: salts composition Hybridizations though are always performed with salt. the presence of monovalent cations (e.g. Na+ ions) stabilizes the duplex, whereas chemical denaturants (such as formamide and urea) destabilize the duplex by chemically disrupting the hydrogen bonds. The formula for the Effective Tm (Eff Tm).  Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(% formamide) Molecular Diagnostics 34

35. 35 Melting temperature (Tm) Tm (°C)Hybrids a Or for other monovalent cation, but only accurate in the 0.01-0.4 M range.a Or for other monovalent cation, but only accurate in the 0.01-0.4 M range. b Only accurate for %GC in the 30% to 75% range.b Only accurate for %GC in the 30% to 75% range. c L = length of duplex in base pairs.c L = length of duplex in base pairs. d Oligo, oligonucleotide; ln, effective length of primer = 2 × (no. of G + C) + (no. of A + T).d Oligo, oligonucleotide; ln, effective length of primer = 2 × (no. of G + C) + (no. of A + T). Note that for each 1% formamide, the Tm is reduced by about 0.6°C, while the presence of 6 M urea reduces the Tm by about 30°CNote that for each 1% formamide, the Tm is reduced by about 0.6°C, while the presence of 6 M urea reduces the Tm by about 30°C 81.5 + 16.6 (log 10 [Na + ] a ) + 0.41 (%GC b ) - 500/L c DNA-DNA 79.8 + 18.5 (log 10 [Na + ] a ) + 0.58 (%GC b ) + 11.8 (%GC b ) 2 - 820/L c DNA-RNA or RNA-RNA For <20 nucleotides: 2 (l n ) For 20 35 nucleotides: 22 + 1.46 (l n ) oligo d -DNA or oligo d -RNAd