Blotting and Detection Methods

1. Blotting and Detection Methods Nucleic Acid Blotting M�rcia A. Duranti Objective of the seminar is to provide technical information about Amersham Pharmacia Biotech�s products for nucleic acid blottingObjective of the seminar is to provide technical information about Amersham Pharmacia Biotech�s products for nucleic acid blotting

2. Overview of the blotting process What are Southern and Northern blots Electrophoresis and membrane transfer Probe labelling Hybridisation Detection options Troubleshooting

3. Southern and Northern blotting

4. Southern and Northern Blotting Southern blot Specific fragments within electrophoretically fractionated DNA detected by hybridization to �labelled� nucleic acid probe(s) Transfer of DNA fragments from an electrophoresis gel to a solid support [Southern, E.M. (1975), J. Mol. Biol. 98, 503-517.] Northern blot Specific RNA species within fractionated RNA detected by hybridization to �labelled� nucleic acid probe(s) Transfer of RNA fragments from an electrophoresis gel to a solid support [Alwine, J.C. et al. (1979), in Methods in Enzymology Vol. 68, Wu, R. (ed.), Academic Press, New York, p.220.] � Note that Southern Blotting refers to the transfer of DNA � DNA is transferred perpendicular to direction of original electrophoresis � Ideally, pattern on membrane is identical to that in gel � Labelling of probe will be addressed later � Specificity obtained by hybridization of complementary sequences according to Watson/Crick base-pairing rules.� Note that Southern Blotting refers to the transfer of DNA � DNA is transferred perpendicular to direction of original electrophoresis � Ideally, pattern on membrane is identical to that in gel � Labelling of probe will be addressed later � Specificity obtained by hybridization of complementary sequences according to Watson/Crick base-pairing rules.

5. Schematic of the blotting process

6. Why Southern Blot? Identity Testing Physical Genetic Mapping Genetic Linkage/Relatedness Diagnosis of Presence of Genetic Markers Presence/Absence of DNA Sequences � Identity Testing: DNA Fingerprinting (forensics, paternity testing, heritage, etc.) � Presence/Absence: screen mutants� Identity Testing: DNA Fingerprinting (forensics, paternity testing, heritage, etc.) � Presence/Absence: screen mutants

7. Why Northern Blot? Level of Expression Comparative Expression Patterns cell type to cell type developmental stages growth conditions RNA Processing/Structural Studies � Since the abundance of a specific message is dependent on the exact conditions at which the preparation was isolated, the level of message can be used as a measure of expression under those conditions � Measurement tends to be comparative and, as such, relative rather than absolute � Can detect transient processing intermediates if sufficiently abundant� Since the abundance of a specific message is dependent on the exact conditions at which the preparation was isolated, the level of message can be used as a measure of expression under those conditions � Measurement tends to be comparative and, as such, relative rather than absolute � Can detect transient processing intermediates if sufficiently abundant

8. Electrophoresis and membrane transfer

9. Choice of Electrophoresis Gels Agarose vs. Acrylamide DNA/RNA fragment size and resolution Gel concentration Resolution, transfer efficiency

10. Gel Treatment Southern blotting Depurination: mild acid treatment of DNA in gel breaks large fragments to facilitate subsequent transfer (optional) Denaturation: alkaline treatment of DNA in gel permits hybridization to probe after transfer Neutralization (optional) Northern blotting Samples can be Total RNA or polyA+ RNA (mRNA) Denaturing electrophoresis conditions glyoxal formaldehyde (rinsed out of gel prior to transfer) Partial alkaline hydrolysis (optional) � If ethidium bromide staining is performed, should be done before subsequent gel treatments � Two choices: transfer DNA & try to denature it after it is bound to membrane or denature DNA in gel and, keeping it denatured, transfer it to membrane where it is bound that way � Large fragments transfer less efficiently so want to introduce occasional breaks (keeping sequence identity and location in electropherogram intact) before transfer � UV light may be as effective but may be more difficult to control. Can also introduce crosslinks that inhibit hybridization. � Usually soak gel in 0.4M NaOH for ~30 minutes to denature fragments in situ � If ethidium bromide staining is performed, should be done before subsequent gel treatments � Two choices: transfer DNA & try to denature it after it is bound to membrane or denature DNA in gel and, keeping it denatured, transfer it to membrane where it is bound that way � Large fragments transfer less efficiently so want to introduce occasional breaks (keeping sequence identity and location in electropherogram intact) before transfer � UV light may be as effective but may be more difficult to control. Can also introduce crosslinks that inhibit hybridization. � Usually soak gel in 0.4M NaOH for ~30 minutes to denature fragments in situ

11. Choice of Membrane Nitrocellulose fragile poor binding of small fragments (<500 bp) limited range of buffer compatibility Nylon higher binding capacity than nitrocellulose higher tensile strength than nitrocellulose positively charged has higher binding capacity than neutral membranes Once the decision has been made to perform a Southern blot, the first choice is to select a type of membrane to �receive� the DNA � All membranes need certain characteristics � They should bind DNA strongly - don�t want it washing away � They should bind as much DNA as presented to them � Depending on how the blot is performed, they need to either allow buffer to pass through and/or allow electric current to flow through � Other contaminating molecules should not bind or interfere with DNA binding � Nitrocellulose was one of the first membranes used � NC worked OK in many applications but had limitations � Fragility especially problematic for re-probing � Activated Paper was used by some people but lost popularity � Claims that Xerox paper works! � Nylon now the membrane of choice � Higher binding capacity � More durable � Greater range of binding (although fragments<50 bp don�t bind well)Once the decision has been made to perform a Southern blot, the first choice is to select a type of membrane to �receive� the DNA � All membranes need certain characteristics � They should bind DNA strongly - don�t want it washing away � They should bind as much DNA as presented to them � Depending on how the blot is performed, they need to either allow buffer to pass through and/or allow electric current to flow through � Other contaminating molecules should not bind or interfere with DNA binding � Nitrocellulose was one of the first membranes used � NC worked OK in many applications but had limitations � Fragility especially problematic for re-probing � Activated Paper was used by some people but lost popularity � Claims that Xerox paper works! � Nylon now the membrane of choice � Higher binding capacity � More durable � Greater range of binding (although fragments<50 bp don�t bind well)

12. Membrane selection Nylon or Nitrocellulose Charged or Neutral (uncharged)

13. Transfer Buffer Southern blotting Neutral High salt (20� SSC) required for binding of DNA to nitrocellulose Nylon permits range of ionic strengths Alkaline (charged nylon membranes) Eliminates gel neutralization step Rapid, efficient transfer Northern blotting Neutral, high salt 10� or 20� SSC 10� SSCP � Different membranes allow (require) different buffers for transfer � Alkaline now preferred but need to use charged nylon--NaOH makes NC very brittle� Different membranes allow (require) different buffers for transfer � Alkaline now preferred but need to use charged nylon--NaOH makes NC very brittle

14. Transfer Methods Capillary blotting traditional (upward) downward Vacuum blotting Electrophoretic blotting(must use electrophoretic buffers) Capillary blotting: This technique is a standard one for subsequent hybridization according to Southern (1975) (Southern blot) during DNA separations. The transfer of RNA on to a covalently binding film or nylon membrane which is now known under the name Northern blot also uses this technique (2).(1) Southern EM. J Mol Biol. 98 (1975) 503-517.(2) Alwine JC, Kemp DJ, Stark JR. Proc Natl Acad Sci USA, 74 (1977) 5350-5354. Vacuum blotting: This technique is mostly used instead of capillary blotting (3). It is important to have a controlled low vacuum with, depending on the case, a 20 to 40 cm high water column to prevent the gel matrix from collapsing. An adjustable mechanical pump is used since a water pump yields a vacuum that is too high and irregular. The surface of the gel is accessible to reagents during the entire procedure. (3) Olszewska E, Jones K. Trends Gen. 4 (1988) 92-94. Electrophoretic blotting: Electrophoretic transfers are mainly used for proteins SDS electrophoresis (4,5). Only in some cases also nucleic acids are transfer with the help of an electric field. (4) Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA. 76 (1979) 4350-4354. (5) Burnette WN. Anal Biochem. 112 (1981) 195-203. Capillary blotting: This technique is a standard one for subsequent hybridization according to Southern (1975) (Southern blot) during DNA separations. The transfer of RNA on to a covalently binding film or nylon membrane which is now known under the name Northern blot also uses this technique (2).(1) Southern EM. J Mol Biol. 98 (1975) 503-517.(2) Alwine JC, Kemp DJ, Stark JR. Proc Natl Acad Sci USA, 74 (1977) 5350-5354. Vacuum blotting: This technique is mostly used instead of capillary blotting (3). It is important to have a controlled low vacuum with, depending on the case, a 20 to 40 cm high water column to prevent the gel matrix from collapsing. An adjustable mechanical pump is used since a water pump yields a vacuum that is too high and irregular. The surface of the gel is accessible to reagents during the entire procedure. (3) Olszewska E, Jones K. Trends Gen. 4 (1988) 92-94. Electrophoretic blotting: Electrophoretic transfers are mainly used for proteins SDS electrophoresis (4,5). Only in some cases also nucleic acids are transfer with the help of an electric field. (4) Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci USA. 76 (1979) 4350-4354. (5) Burnette WN. Anal Biochem. 112 (1981) 195-203.

15. Capillary Blotting Diagram of classic Southern setup. For long transfers, the blotting material may need to be replaced when it nears saturation.Diagram of classic Southern setup. For long transfers, the blotting material may need to be replaced when it nears saturation.

16. Vacuum Blotting Gentle vacuum source used to pull liquid through the gel, DNA moves with the buffer Decreases transfer time required Agarose only

17. Vacuum Blotting

18. Electrophoretic Transfer Decreases transfer time required Used with acrylamide or agarose Requires low ionic strength buffer Requires nylon membranes High salt buffers required for binding to NC are too conductive for electro-transfer. High currents overheat the gel. Nylon membranes bind DNA at low ionic strength.High salt buffers required for binding to NC are too conductive for electro-transfer. High currents overheat the gel. Nylon membranes bind DNA at low ionic strength.

19. Membrane Fixation Bake in oven (neutral transfers) Vacuum baking for nitrocellulose UV crosslinking (uncharged nylon) Alkaline transfer - buffer rinse/air drying needed for charged nylon membranes Not recommended for RNA After fragments are transferred, need to covalently bind DNA to membrane. Original oven protocols require at least 2 h at 80 C for NC blots. UV cross linkers designed for this task give reproducible dose of energy and take only seconds For Alkaline transfer, only need to rinse residual NaOH from membrane (so that it doesn�t interfere with subsequent hybridization) then dry membrane to permanently fix DNA on membraneAfter fragments are transferred, need to covalently bind DNA to membrane. Original oven protocols require at least 2 h at 80 C for NC blots. UV cross linkers designed for this task give reproducible dose of energy and take only seconds For Alkaline transfer, only need to rinse residual NaOH from membrane (so that it doesn�t interfere with subsequent hybridization) then dry membrane to permanently fix DNA on membrane

20. Probe labelling techniques

21. Factors affecting label choice Type of Detection method Sensitivity required Probe Type Ease-of-use

22. Probe Type Long Probes - highest sensitivity 1 kb vs 10 kb probe Southern/Northern Blots Mid-size Probes - intermediate sensitivity plasmid inserts (< 1 kb) PCR fragments Oligonucleotide Probes - lower sensitivity Screening Applications

23. Types of Label INDIRECT biotin/streptavidin hapten/antihapten Ig IgG/antispecies IgG

24. Enzyme Labelling Strategies DIRECT Enzyme linked directly to probe HRP - horseradish peroxidase Alkaline phosphatase INDIRECT Probe labelled with tag (hapten); enzyme is introduced later Random prime 3�-Oligo tailing RNA labelling

25. AlkPhos Direct labelling Based on patented Renz technique Thermostable AlkPhos Formaldehyde cross-linker Thirty minute reaction Scaleable protocol Probes stable for at least six months

26. AlkPhos Direct - summary Unique product for high sensitivity detection Save hours over haptenated systems Lower backgrounds than hapten systems Based on successful ECL direct

27. Hybridisation

28. Hybridisation to Immobilized Targets Pre-hybridization blocking of membrane sites to prevent non-specific probe binding prepare membrane for hybridisation conditions Hybridisation sensitivity specificity Stringency Washes

29. Factors Affecting Rate of Hybridization Probe Concentration/Complexity Duplex Length Base Composition Temperature Ionic Strength Destabilizing Agents (formamide, urea, etc.) Mismatched Base Pairs Viscosity

30. Factors Affecting Hybrid Stability Mismatched Base Pairs Ionic Strength Destabilizing Agents Duplex Length Base Composition

31. Probe concentration Radioactive applications 2-5ng/ml final volume gives best results 0.5 - 1.5ng/ml if using rate enhanced buffers Non-radioactive applications 5-10ng/ml in non-radioactive applications 1-5ng/ml for colonies plaques

32. Factors Affecting Hybridization Factors Affecting Sensitivity Probe Specific Activity Amount of Target DNA or RNA Type of Label Factors Affecting Specificity Homology between probe and target Wash stringency ionic strength temperature

33. Factors Affecting Hybridization Temperature Vary with probe Low Temperature - Lower stringency : ? especificity Long Probes (< 100 bases) : 65-68 oC Short/Oligo Probes (<50 bases) : Tm - 5 oC Tm = (4x no. G+C bases) + (2x no. A+T)

34. Stringency Washes Ionic strength - Temperature Low stringency : high salt stabilizes mismatched sequences High salt, Low temperature Medium stringency Medium salt, Medium temperature High stringency : low salt only compatible with good matches Low salt, High temperature

35. Stringency control via temperature

36. Detection options

37. Detection Scheme Comparison

38. Ultimate detection sensitivities

39. Relative light profiles of chemiluminescent systems

40. Speed of detection

41. Radioactive resolution & sensitivity

42. Mechanism of Alkaline Phosphatasemediated dioxetane chemiluminescence

43. CDP-Star detection

44. Northern blotting

45. Chemifluorescence Detection

46. Data Acquisition & Analysis Radioactivity X-ray film (Autoradiography) Film Scanners Phosphorimagers Fluorescence/Chemifluorescence Chemiluminescence X-ray film (Autoradiography) Film Scanners CCD Cameras/Scanners

47. Radioactive Detection Autoradiography Requires darkroom and developing protocol Film consumption Isotope flexibility limited with extra materials Narrow dynamic range Flexible exposure range Quantitation requires scanning densitometer Phosphorimager Requires expensive instrumentation Can reuse storage phosphor cassette Wide range of isotopes Broad dynamic range Flexible exposure range Accurate quantitation of data directly

48. Chemiluminescent Detection Autoradiography Requires darkroom and developing protocol Film consumption Narrow dynamic range Flexible exposure range Quantitation requires scanning densitometer Low cost option CCD Camera/Scanner Requires expensive instrumentation; CCD cooling Some scanners can use storage phosphor cassettes (with limited sensitivity) Broad dynamic range Flexible exposure range Accurate quantitation of data directly

49. Troubleshooting

50. Troubleshooting Poor transfer With capillary and vacuum transfer of agarose gels, gel pores can become collapsed if liquid is drawn from the gel. DNA becomes trapped inside gel matrix. Poor binding or contact with the membrane Insufficient transfer time Larger fragments or thicker gels require longer transfer

51. Troubleshooting No or weak detection Detection chemicals Use fresh reagents Poor binding to membrane Old nitrocellulose Try charged nylon Poor transfer Stain gel Stringency too high Reduce temperature increase salt concentration

52. Troubleshooting High background Insufficient blocking of membranes Inadequate washing Bacterial contamination of buffers Try nitrocellulose instead of nylon Bad probe Expired membranes