4. DNA HYBRIDIZATION TECHNIQUES Flashcards
- It is a process of establishing noncovalent and sequence specific interaction between two or more complementary strands of nucleic acid into a single hybrid
HYBRIDIZATION
are synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing.
DNA PROBE / GENE PROBE
Hybridization technology that is used for Gene structure
Southern blot
Hybridization technology that is used for Transcript structure, processing, gene expression
Northern blot
Hybridization technology that is used for Protein processing, gene expression
Western blot
Hybridization technology that is used for DNA-binding proteins, gene regulation
Southwestern blot
Hybridization technology that is used for Modification of western blot using enzymatic detection (PathHunter); also, detection of specific agriculturally important proteins
Eastern blot
Hybridization technology that is used for Transfer of high-performance liquid chromatography (HPLC)-
separated lipids to polyvinyl difluoride (PVDF) membranes for analysis by mass spectrometry
Far-eastern blot 29,30
Southern blot target
DNA
Northern blot target
RNA
Western blot target
Protein
Southwestern blot target
Protein
Eastern blot target
Protein
Far-eastern blot 29,30 target
Lipids
Southern blot probe
Nucleic acid
Northern blot probe
Nucleic acid
Western blot probe
Protein
Southwestern blot probe
DNA
Eastern blot probe
Protein
Far-eastern blot 29,30 probe
None
Who is the Southern blot named after?
Edwin Southern
What is the first step in the Southern blot procedure?
Isolate and cut genomic DNA with restriction enzymes
How are DNA fragments separated in Southern blotting?
Gel electrophoresis
What is done to the DNA fragments after separation? [southern blot]
They are denatured and transferred to a solid support
Example of solid support used for southern blot after DNA fragments are denatured
nitrocellulose
What probe is used to detect DNA fragments?
Labeled probe (complementary DNA or RNA)
How is the presence of a DNA sequence determined? [southern blot]
By detecting the signal from the probe
What was the original method of detection in Southern blotting?
Radioactively labeled probe
What types of DNA regions are analyzed using Southern blot?
Large regions (10 kb to over 100 kb)
What has replaced Southern blot for many applications?
PCR (for many, but not all, applications)
What does Southern blot analyze at the molecular level?
Any gene or gene region in prokaryotes or eukaryotes
What is Southern blot still commonly used for?
Characterization of large DNA regions
What is the first step in the Southern blot procedure?
Digestion of test DNA with restriction enzymes.
What determines the choice of restriction enzymes in Southern blotting?
Genetic locus and application.
What is the result of incomplete restriction enzyme cutting in Southern blotting?
anomalous patterns
How are DNA fragments resolved after restriction enzyme digestion in Southern blotting?
Gel electrophoresis.
What determines the percentage and type of gel used in Southern blotting?
DNA fragment size.
What should be run alongside test samples during electrophoresis in Southern blotting?
Molecular-weight standard
How are large DNA fragments (10,000-20,000 bp) best resolved in Southern blotting?
0.7% agarose gel, low voltage, long run.
What does a smear on an ethidium bromide-stained gel illuminated with UV light indicate during Southern blotting?
Complete digestion.
What does a large aggregate of DNA near the top of the gel lane indicate in Southern blotting?
Incomplete digestion.
What does a smear primarily located in the lower region of the gel lane indicate in Southern blotting?
DNA degradation.
What should be done if uncut or degraded DNA is present after electrophoresis in Southern blotting?
Repeat digestion or purify DNA.
How much genomic DNA is typically required for each restriction enzyme digestion in Southern blotting?
10-50 µg.
What should be done if multiple restriction enzymes are used for Southern blotting?
Digest samples separately.
How long is the typical digestion time for complete DNA cutting in Southern blotting?
3+ hours.
What is the goal of the Southern blot procedure?
Analyze specific DNA region.
How are target DNA fragments detected in Southern blotting?
Hybridization with complementary probe.
What must happen to double-stranded DNA fragments before hybridization in Southern blotting?
Denaturation.
is used to prepare larger DNA fragments (over 500 bp) by removing purine bases, making them easier to denature.
Depurination
What is used to depurinate DNA fragments in Southern blotting?
Dilute HCl
How is DNA denatured in Southern blotting?
Exposure to sodium hydroxide (NaOH).
What is the role of sodium hydroxide in denaturation?
Breaks hydrogen bonds.
Common membranes used in Southern blotting?
Nitrocellulose,
nylon,
cellulose modified with:
diethyl aminoethyl,
carboxymethyl (CM).
are used for immobilizing proteins for probing with antibodies
(western blots).
polyvinyl difluoride (PVDF)
Nitrocellulose-based membranes bind ____________ of nucleic acid per square
centimeter
70 to 150 μ g
Membrane pore sizes that are
suitable for DNA fragments from a few hundred bases up to those greater than 20,000 bp in length.
0.05 to 0.45 μ m
has a high binding capacity for proteins as well as nucleic acids. It is the most versatile medium for molecular transfer applications. It is also
compatible with different transfer buffers and detection systems
Pure nitrocellulose
Membrane used in southern blot that is more appropriate for applications where multiple probings may be necessary
Reinforced nitrocellulose
These membranes can be formulated with a net neutral charge to decrease nonspecific binding when performing southern blot .
Mechanically stable membranes
A covalent attachment of nucleic acid to these membranes [southern blot] is achieved by exposure of the DNA on the membrane with _____________
UV-light cross-linking
Are more effective when binding small fragments of DNA.
These membranes, however, are more likely to retain protein or other contaminants that will contribute to background noise after the membrane is probed.
Membranes with a positive charge
What transfer method did southern use in order to transfer the DNA sample from the gel to the nitrocellulose membrane
Capillary transfer
What types of buffers can be used during capillary transfer to facilitate DNA movement?
10 × saline sodium citrate (10X SSC) or
commercially available transfer buffers.
What should be avoided during the transfer to ensure effective binding of DNA to the membrane?
Folding or creasing of the membrane.
What type of cellulose effectively binds nucleic acids and negatively charged proteins?
Diethylaminoethyl (DEAE)-conjugated cellulose.
What types of membranes are used specifically for protein (western) blotting?
PVDF and charged carboxymethyl cellulose membranes.
How do PVDF and charged carboxymethyl cellulose membranes bind nucleic acids and proteins?
Hydrophobic and ionic interactions.
What is the binding capacity range for PVDF membranes?
20 to 150 μg/cm².
Types of transfer methods
Capillary transfer
electrophoretic transfer
Vacuum transfer
electrophoretic transfer two types
Tank
Semidry
A simple and inexpensive method that requires no instruments, but can be less than optimal, especially with large gels. Bubbles and salt crystals between the membrane and gel can lead to information loss or staining artifacts. The procedure can take from a few hours to overnight for large fragments.
Capillary transfer
This method uses electrodes placed above and below the gel, transferring electric current through the gel and membrane using electrophoresis buffer, allowing the DNA to move to the membrane.
Tank method.
In this approach, electrodes contact the gel-membrane sandwich directly, only requiring enough buffer to soak the gel and membrane. This method is often used for smaller proteins.
Semidry method.
This method is preferred for transferring large proteins resolved on acrylamide gels, utilizing a tank system for effective transfer.
Tank electrophoretic transfer.
This blotting technique employs suction to facilitate the movement of DNA from the gel to the membrane, allowing for a quicker transfer time of 2 to 3 hours and preventing air trapping issues.
Vacuum transfer.
What methods can be used to permanently immobilize cut, denatured DNA to the membrane after transfer?
Baking in a vacuum oven (80°C, 30 to 60 minutes)
UV cross-linking
Process where we incubate the membrane in a buffer solution without the probe to block nonspecific binding.
prehybridization
What are the key components of prehybridization buffer?
Denhardt solution
salmon sperm DNA,
and sodium dodecyl sulfate (SDS).
Denhardt solution components
Ficoll,
polyvinyl
pyrrolidine,
bovine serum albumin)
Blocking agents specially used for RNA probes
formamide
The membrane is exposed to the prehybridization buffer at the optimal hybridization temperature for
30 minutes
to several hours
a modification of the Southern blot technique, was designed to investigate
RNA structure and quantity
northern blot
Analysis of RNA structure and quantity indirectly reveals mutations in the
regulatory or splicing
signals in DNA.
Agarose concentrations of ____________ are usually employed for northern blots
0.8% to 1.5%
These gels may also be used, especially for smaller transcripts—for instance, for analysis of viral gene expression in northern blot
Polyacrylamide gels
In northern blot procedures After electrophoresis, representative lanes can be cut from the gel, soaked in ________ to remove the denaturant
ammonium acetate
Northern blot makes use of the stains ______________
to assess quality and equivalent sample loading
acridine orange or ethidium bromide
Denaturant such as formaldehyde must be removed from the gel before transfer because it inhibits binding
of the RNA to nitrocellulose. This is accomplished by rinsing the gel in
deionized water
During northern blot procedures, If the RNA has been denatured in glyoxal, the
membrane must be soaked in warm ______________
to remove any residual denaturant immediately before prehybridization
Tris buffer (65°C)
There are many variations on western blots.
Generally, serum, cell lysate, or extract is separated on which type of gels
SDS-polyacrylamide gels (SDS-PAGE) or isoelectric focusing gels (IEF)
What does SDS-PAGE resolve proteins by?
Molecular weight
What does IEF resolve proteins by?
Charge
Which chemicals may be used to separate proteins into subunits in a Western blot?
Dithiothreitol or 2-mercaptoethanol.
What are the typical concentrations of polyacrylamide used in Western blot gels?
5% to 20%
How much protein is typically loaded per well for a Western blot?
1 to 50 μg
What treatment is applied to samples before loading in a Western blot?
Samples are treated with denaturants, such as 0.04 M Tris HCl, pH 6.8, 0.1% SDS.
What can denaturing gels affect in Western blotting?
They can affect epitopes, preventing binding with antibodies.
How can proteins be renatured before transfer in Western blotting?
By pretreating gels with 20% glycerol in 50 mM Tris-HCl, pH 7.4.
What transfer methods are used for proteins in Western blotting?
Capillary or electrophoretic transfer.
What blocking agent is used to prevent antibody binding to the membrane when performing western blot
0.1% Tween 20 in 0.05 M Tris, 0.15 M NaCl, and 5% dry milk.
How are proteins likely bound to nitrocellulose membranes?
Hydrophobically
What other membrane types are used for protein blotting?
PVDF, DEAE-cellulose (anion exchange), and CM-cellulose (cation exchange).
How long is the membrane incubated with the primary antibody in Western blotting?
12 to 16 hours.
How is the signal developed in Western blotting?
With the addition of a chemiluminescent or colorimetric substrate.
