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- Electrophoresis2- Transfer3- Blocking 4- Probing5- Detection6- 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 Glass plates Whatman paper 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 Gel Recirculating buffer 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 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  2 o Ab with a label will bind to 1 o 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  32 P is commonly used as a label  Emits radiation that can be easily detected by autoradiography  Nucleotides that incorporate 32 P 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- Molecular Weight 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 Controls Positive 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  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 Isotope Non-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 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 of DNA appear as a smear cassette filter DNA extraction DNA digestion Gel electrophoresis Blot dismantled Hybridisation: Stringency washes Autoradiography Disease gene Gel in NaOH Southern blot Radioactive probe added to filter Nylon filter Paper towels Gel 10x SSC Chromatography paper 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 Isotope Non-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 1 o Ab, washing Addition of 2 o 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)

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