introduction definition a technique by which a macromolecule such as dna, rna, or protein is...
TRANSCRIPT
Introduction
Definition•A technique by which a macromolecule such as DNA, RNA, or protein is resolved in a gel matrix, transferred to a solid support, and detected with a specific probe.
•Used to identify specific molecules in a complex mixture of related molecules.
Introduction
Common techniques include: ○Southern blotting (DNA) ,
○Northern blotting (RNA) ,
○and immunoblotting (for protein; also known as Western blotting).
The blotting procedures can be divided into six main steps
1- Electrophoresis 2- Transfer 3- Blocking
4- Probing 5- Detection 6- Results
1- Electrophoresis
The molecule of interest is present in a complex mixture of molecules.
Separate on the basis of size.
Separating the molecules by gel electrophoresis on either: an agarose
or polyacrylamide gel.
Gel Electrophoresis
2- Transfer (blotting)
Following separation, the molecules are transferred to a solid support such as:
a nylon,
nitrocellulose,
or polyvinylidene fluoride (PVDF) membrane.
Carbon copy of the molecules that were present in the gel are now immobilized on a membrane.
The membrane of Choice
The membrane of choice is determined by the sensitivity required and the detection method to be used.
Type of Membrane
2- Transfer (blotting)- Blotting Types
2- Transfer (blotting)- Capillary Transfer Fragments are eluted from the gel and deposited onto
the membrane by buffer that is drawn through the gel by capillary action.
Buffer
Wick (filter paper) Filter paper
Agar gel with DNA
Membrane
Weight
Paper towel stack
- +
Buffer Buffer
Glass plates
Whatmanpaper
Nitrocellulose filter
Gel
2- Transfer (blotting)-
Electrophoretic Transfer• The negatively charged nucleic acid molecules will
move from the gel to the membrane
Electroblotting
Nitrocellulose filter
Porous plate
GelRecirculatingbuffer
Vacuum
2- Transfer (blotting)- Vacuum Transfer• Nucleic acids are eluted by buffer that is drawn through
the gel by application of negative pressure (a vacuum).
2- Transfer (blotting)- Cross-Linking Once transferred to a membrane, they
have to be linked to the membrane.
UV irradiation, covalently attach the nucleic acids to the membrane
Covalent bond between the amide groups on the nylon and the carbonyl groups found on the thymine and uracil bases
2- Transfer (blotting)- Cross-Linking Alternatively, the membrane can be baked
at 80◦C for 2 hr.
Dehydration of the nucleic acids on the blot, resulting in the generation of stable
hydrophobic interactions between the nucleic acid and the membrane.
3- Prehybridization (Blocking) The transferred nucleic acids only occupy a
limited amount of the surface area of the membrane.
The molecules in the prehybridization solution coat the rest of the membrane.
In the absence of such a treatment, the probe would: associate with the unoccupied sites on the membrane,
resulting in very high background and a very low signal-to-noise ratio.
4- Probing
Membrane is now incubated with a specific probe that binds to the protein or nucleic acid sequence of interest.
For southern or northern, a fragment of DNA of variable length (usually 100-1000 bases long)
4- Probing
The probe will have two properties: First, anneal specifically with the
sequence of interest.
Second, modified in such a way as to allow for the detection of the annealed sequences.
4- Probing
Probe used for an immunoblot is an antibody that recognizes a particular protein
2o Ab with a label will bind to 1o Ab with high affinity
Unbound probe or nonspecifically bound probe is removed by washing the membrane
4- Probing
Probe
4- Probing- Production of Probes The availability of a gene probe is essential
in many molecular biology techniques.
The information needed to produce a gene probe may come from many sources,
e.g. genetic databases.
Genbank and EMBL search to identify particular sequences relating to a specific gene or protein.
Use related proteins from the same gene family to gain information DNA sequence.
Similar proteins or DNA sequences but from different species may also provide a starting point with which to produce a probe.
4- Probing- Production of Probes
4- Probing- Labeling of Probes To visualize DNA or RNA, the nucleic acid
should to be attached to a label:○ radioactive,
○ colored,
○ fluorescent,
○ Or luminescent.
The three main choices are:
A. radioisotopes,
B. fluorophores,
C. and small-molecule binding partners
4- Probing- Labeling of Probes- A-
Radioisotopes 32P is commonly used as a label
Emits radiation that can be easily detected by autoradiography
Nucleotides that incorporate 32P are commercially available.
Can be readily incorporated into DNA by enzyme-catalyzed reactions.
4- Probing- Labeling of Probes- B- Fluorophores Fluorophores are molecules that
absorb light at one wavelength and then emit light at a different wavelength.
Incorporate fluorophores: chemically during DNA synthesis,
or enzymatically
4- Probing- Labeling of Probes- C- Small molecule binding partners Small organic molecules that are
recognized by:antibodies or
other protein binding partners.
Common molecules are biotin and digoxigenin
5- Detection
Streptavidin covalently conjugated with a detection moiety.
For example, streptavidin conjugated directly to a fluorophore or to enzymes such as horseradish peroxidase or alkaline phosphatase.
Enzymes detected by their action on provided substrates that deposit products which are colored, luminescent, or fluorescent.
4- Probing- Labeling of Probes- C- Small molecule binding partners
Membrane
Hybridization
Probe is generated and added to the blot for 1 to 24 hr.
Time to hybridize the blot depends on a variety of factors and must be determined empirically.
Overnight to maximize hybridization of the probe to the target
5- Detection
To visualize the bound probe.
Determined by the nature of the probe.
If a radioactive probe, autoradiography exposure of the blot to X-ray film will
allow for detection and quantitation of the bound probe.
5- Detection
If chemical- or enzyme-based, substrates are added
the resulting signal is developed
and can be documented by: ○ colorimetric,
○ or chemiluminescent imaging.
5- Detection
For fluorescently labeled nucleic acid use imaging equipment to excite the
fluorophore
And the appropriate filter to detect the emitted light.
6- Results and Analysis Once the blot is developed, the resulting
banding pattern can be analyzed.
Analysis involves: determining the amount and molecular weight or size
of the molecules on the blot
and comparing the results to the predicted pattern.
To determine the molecular weight a standard curve of size versus migration distance is derived from the molecular weight markers
Distance (mm)
Log
- M
ole
cula
r W
eig
ht
1
2
3
Best Fit Line
Positive & Negative Controls
Negative controls
Include samples that are identical to the experimental sample but are missing the target that the probe is supposed to recognize.
Very useful in determining the existence of any background that can be due to cross-reactivity between the probe and the sample.
Positive & Negative ControlsPositive control
Include samples that contain the protein or nucleic acid of interest.
When included in the experiment allows the investigator to confirm that the experiment was successfully executed.
No signal indicates that the problem lies with the experimental samples and not with the procedure.
Southern Blotting
Developed by E.M. Southern in 1975.
A technique used in molecular biology to check for the presence of a particular DNA sequence in a DNA sample.
Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer & Fixing DNA onto membrane
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
IsotopeNon-isotope
Preparing the samples and running the gel
Extraction of DNA
DNA must first be fragmented into small pieces that can migrate through an agarose gel matrix.
Restriction enzymes are used to fragment the DNA
Preparing the samples and running the gel
DNA Digestion Restriction enzymes recognize
specific DNA sequences in DNA and cleave the DNA at these restriction sites.
Digestion with a given restriction enzyme produces a set of fragments that are easily separated by agarose gel electrophoresis.
The enzyme EcoRI cutting DNA at its recognition sequence
Preparing the samples and running the gel
DNA Digestion
Nucleic acids are negatively charged at a neutral pH
This allows their migration through an electric field
Agarose is a highly porous polysaccharide that acts as a sieve, allowing the fragments of DNA to be separated according to length.
Preparing the samples and running the gel
Electrophoresis
Preparing the samples and running the gel
Electrophoresis
Preparing the samples and running the gel
Denature the DNA Denature DNA with an alkaline
solution such as NaOH.
Double stranded becomes single-stranded.
Single strands are ready to be transferred to a solid support
Southern Transfer & Fixing DNA
Transfer the DNA from the gel to a solid support.
Baking the membrane at 80°C for 2 h in a vacuum oven.
Or expose to ultraviolet
Probe Preparation, Prehybridization & Hybridization A labeled probe is prepared which is
complementary for the sequence we are looking for
Prehybridization to block sites where probe can bind on the membrane
Hybridization
Post-hybridization washing
Following hybridization, the blot must be washed to remove unassociated and nonspecifically annealed probe from the blot.
Detection
Steps in Southern Blotting
Denaturation of patient’s DNA in gel
Fragments ofDNA appearas a smear
cassettefilter filter
DNA extraction
DNA digestion
Gel electrophoresis
Blot dismantled
Hybridisation:Stringency washes
Autoradiography
Disease gene
Gel inNaOHSouthern blot
Radioactive probeadded to filter
Nylon filter
Paper towels
Gel
10x SSC
Chromatographypaper support
X ray film
Identify mutations, deletions, or rearrangements that alter the integrity of a specific gene, useful in the prognosis of certain types of
cancer
Tool for molecular cloning, providing a mechanism for localization of specific sequences
Uses of Southern Blotting Technique
The DNA blot can also be used to assess the relative copy number of a specific gene. Useful in detecting gene amplification.
Southern blotting may be used to confirm the specificity of the test reaction product.
To search for a homologous gene different organisms
Uses of Southern Blotting Technique
Southern Blotting as a Diagnostic Method Restriction fragment length polymorphism
(RFLP) analysis was one of the early methods to diagnose point mutations implicated in genetic diseases
The change in the size of detected fragments with a gene-specific probe signals the presence of mutation in the analyzed gene
Has been applied to the diagnosis of hemophilia A, Sickle cell anemia and others
Southern Blotting as a Diagnostic Method
PCR has replaced the Southern blotting
Cystic fibrosis, Duchenne muscular dystrophy, sickle cell anemia thalassaemia, and others, are now diagnosed by polymerase chain reaction (PCR).
Genomic and Plasmid DNA Analyses
Does a particular genomic locus or region of plasmid DNA contain a sequence of interest? Where does it reside?
Techniques: Restriction enzyme digestion
Agarose gel electrophoresis
Southern blot
Genomic and Plasmid DNA Analyses
How many genomic loci contain a particular sequence of interest, or how many copies of that sequence does a genome contain?
Technique:Southern blot
The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
IsotopeNon-isotope
Allows identification of specific messenger RNA sequences within a mixture of RNA molecules.
The final signal achieved on the blot is proportional to the number of specific sequences present, allowing for a quantitative analysis of gene
expression.
Differences between Southern & Northern
RNA rather than DNA is separated by size on gel
Cutting by nucleases before electrophoresis unnecessary.
Although RNA is single stranded, it has a tendency to bend back on itself and form base-paired loops, hairpins, and other secondary structures.
Denaturing agents (e.g., formamide) must be added to the electrophoresis buffer to prevent the formation of secondary structures
Differences between Southern & Northern
RNA Paranoia
RNA paranoia is very important from start to finish.
The work area should be cleaned with RNase inhibitors.
Gloves should be changed if non-RNase-free items have been touched (e.g., your hair, your face, your arm, notebook paper).
Uses of Northern Blotting Northern blots can be used to assess
different levels of expression from a particular gene.
For defining post-transcriptional modification such as:
splicing and poly(A) addition,
Gene Expression (Transcription) Analyses
What is the size of a specific gene transcript?
Technique:Northern blot
Gene Expression (Transcription) Analyses
Is a gene of interest expressed (transcribed)?
Technique:Northern blot
Gene Expression (Transcription) Analyses
Is transcription of a gene altered (increased or decreased) under different conditions?
Technique:Northern blot
Real-time PCR (for more quantitative comparison)
The Flow Chart Of Immunoblotting
Electrophorese samples
Transfer proteins from gel to membrane
Blocking
Addition of 1o Ab, washing
Addition of 2o Ab, washing
Detection
Uses of Immunoblotting
Immunoblotting is used to identify specific protein in a mixture
Uses of Immunoblotting
Dot and slot blots
Provide a quick and simple way to determine the amount of an antigen in a sample without performing electrophoresis first.
Proteins are deposited onto the membrane
Probe with the same chromogenic or luminescence protocol as the western blot.
Dot and slot blots
Provides a mean of measuring the abundance of specific proteins without the need for gel electrophoresis,
It does not, however, provide information regarding the size of the fragments.
Proteins
In which cellular structures or organelles do specific proteins reside?
Techniques:Cell fractionation
Immunoblotting
Proteins
What is the molecular mass of a specific protein? Is it post-translationally modified?
Technique:Immunoblotting
Example for Uses of Blotting Techniques Suppose a student was studying a newly
identified gene, X, from cows.
The student then asks three basic questions as part of a research project:
1. Do sheep also have gene X on their chromosomes?
2. Do cows express gene X in their brain tissue?
3. Is the protein product of gene X found in the cow's blood plasma?
Blotting experiments can answer all three of these questions.
Do sheep also have gene X on their chromosomes?
A Southern (DNA) blot will answer the first question.
DNA from a sheep and performed the Southern blotting technique with a probe complementary to that gene.
If the sheep's DNA also contains gene X, there should be a fragment on the nitrocellulose
In other words, the labeled probe will bind to any fragment from the blotted sheep DNA that contains gene X, allowing the student to detect the presence of gene X in sheep.
Do cows express gene X in their brain tissue?
To answer the second question, a Northern (RNA) blot would be used.
The student would isolate RNA from the cow's brain tissue and run it out on the gel.
The same DNA probe used for Southern would then be used to detect whether the RNA that represents gene X expression is present in the brain.
Is the protein product of gene X found in the cow's blood plasma?
To answer the third question, the student would use a Western (protein) blot.
This requires the use of an antibody that specifically reacts with the protein coded for by gene X.
The student first obtains plasma from the cow and uses standard biochemical techniques to isolate the proteins for analysis.
These proteins can then be run out on a gel and transferred to nitrocellulose.
The proteins can then be probed with the labeled antibody.
If the product of gene X is in the plasma, it will bind with the labeled antibody and can thus be detected.
References
Current Protocols Essential Laboratory Techniques (2008)
Molecular Diagnostics (2006)
Medical Biomethods Handbook (2005)
Thank You