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Overview of the blotting process. What are Southern and Northern blotsElectrophoresis and membrane transferProbe labellingHybridisationDetection options Troubleshooting. . Southern and Northern blotting. Southern and Northern Blotting. Southern blotSpecific fragments within electrophoreticall
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1. Blotting and Detection Methods Nucleic Acid Blotting
Mărcia 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