Western and Southern blotting Easy Language Biotechnology 6th Sem

Title: "Southern and Western Blotting Techniques: A Detailed Overview"

Description: This presentation provides an in-depth yet accessible explanation of two essential molecular biology techniques, Southern blotting and Western blotting, commonly used in genetic research, forensic analysis, and protein studies. Ideal for B. Pharm 6th-semester students, this content has been tailored to simplify complex concepts while maintaining the scientific accuracy necessary for professional learning.

Slide Breakdown:
Introduction to Blotting
The basics of molecular blotting techniques are introduced, outlining their purpose in detecting specific DNA, RNA, or proteins from a mixture. A clear analogy to a "book with highlighted passages" is used to make it relatable.

Southern Blotting Overview
Detailed steps of Southern blotting are explained, starting from DNA extraction to visualization. Key concepts such as gel electrophoresis, membrane transfer (using nylon or nitrocellulose), and hybridization with a probe are broken down into simple steps. A particular focus is placed on understanding the role of the DNA probe, using analogies like "highlighter" or "magnet."

The Role of a Probe in Southern Blotting
Special attention is given to explain how probes are designed and used to detect specific DNA sequences in samples. Examples include forensic analysis where probes are used to identify criminal DNA sequences by matching them against evidence found at a crime scene.

Applications of Southern Blotting
This slide explores the wide applications of Southern blotting, ranging from gene discovery and DNA fingerprinting to applications in diagnosing genetic disorders.

Western Blotting Overview
A clear and step-by-step explanation of Western blotting, focusing on how it is used to detect specific proteins in a mixture. The slide covers important steps like protein separation via SDS-PAGE, transfer to a membrane, and antibody binding.

The Role of Antibodies in Western Blotting
This section delves into how primary and secondary antibodies are used to detect proteins. The slide emphasizes the importance of antibodies binding to their target protein and how enzymes linked to secondary antibodies help generate visible signals.

Comparison Between Southern and Western Blotting
A comparative chart shows the main differences between these two techniques, including the target molecules (DNA vs. protein), key reagents (probe vs. antibodies), and the type of signals used to visualize the results.

Forensic Case Study
A real-life crime scene example is used to demonstrate how Southern blotting is applied in forensic science. The DNA from suspects is compared to evidence using DNA probes, which are designed to match specific sequences.

Western Blotting in Medical Diagnostics
A brief look at how Western blotting is critical in diagnosing conditions like HIV, where proteins from the virus are detected in patient samples.

1. Blotting Techniques: Unveiling Genetic and Protein Secrets Explore the powerful techniques of Southern, Northern, and Western blotting for studying DNA, RNA, and proteins. by Rohit Grover

2. The Molecular Foundations 1 DNA DNA is like the instruction manual for all living organisms. It’s a molecule made up of smaller units called nucleotides , which form a long, twisted ladder-like structure (called a double helix). Each DNA molecule contains genes, which are instructions for building proteins and performing other tasks in the cell. 2 RNA Converts DNA instructions into proteins. 3 Proteins Vital molecules that perform essential functions.

3. Southern Blotting: Detecting DNA What is Southern Blotting? Southern blotting is a laboratory technique used to detect a specific DNA sequence in a sample. The process involves cutting DNA into fragments, separating them by size using gel electrophoresis, and then transferring (blotting) these fragments onto a membrane. A labeled DNA probe is then used to bind to the target sequence, allowing for its detection and visualization. This method is often used for identifying genetic mutations, DNA fingerprinting, and studying gene organization. It was named after the scientist Edwin Southern , who developed the technique in 1975. 1 DNA Extraction Extract DNA from the sample. 2 DNA Digestion Cut DNA into fragments. 3 Gel Electrophoresis Separate DNA by size. 4 Transfer to Membrane Blot DNA onto a membrane. 5 Hybridization Use a DNA probe to find target. 6 Detection Visualize the labeled DNA.

4. Southern Blotting in Action Genetic Mutation Detect mutations like sickle cell anemia. Visualization A band indicates the presence of the target.

5. 1. Cutting DNA into Fragments (Restriction Digestion) DNA in its natural state is a long molecule , like a big, tightly packed book with millions of letters. When you want to find a specific section of this DNA), you need to break it into smaller pieces so it's easier to handle. This is done using restriction enzymes . Restriction enzymes are special proteins that recognize specific sequences of DNA and cut the DNA at those spots, like molecular scissors. For example, one enzyme might always cut DNA at a sequence like "GAATTC." After you cut the DNA, you get a bunch of fragments of different sizes, depending on where the enzyme made the cuts.

6. 2. Separating DNA Fragments (Gel Electrophoresis) Once the DNA is cut into smaller pieces, the next step is to separate these fragments by size . This is done using a technique called gel electrophoresis . Gel electrophoresis is like running DNA through a jelly-like substance called agarose gel . You place the DNA fragments in small wells (holes) at one end of the gel, and then apply an electric current . Since DNA has a negative charge, it will move toward the positive side of the gel. Smaller fragments move faster through the gel, while larger fragments move slower . As a result, the DNA fragments get separated based on their size. After the gel electrophoresis is done, you’ll have all the DNA fragments arranged by size in the gel. This forms what’s called a DNA ladder —a pattern of bands that represents different-sized pieces of DNA. After this they undergo strong alkali treatment which will conver double stranded dan into Single stranded dna Alkaline Treatment

7. 3. Transferring DNA to a Membrane (Blotting) Now, you need to move the separated DNA from the gel onto a membrane (which is like a piece of paper or a filter). This membrane will be easier to handle and allows for further testing. To transfer the DNA, you place the gel on top of a special nylon or nitrocellulose membrane . Then, you put layers of paper towels or sponges on top and apply pressure. And buffer used is Saline Citrate Sodium Buffer Capillary action (similar to how water moves up a paper towel) causes the DNA fragments to move out of the gel and stick to the membrane. ( High Water Potential – Low Water Potential ( paper + ve charge dna – ve charge) Why is this step important? Imagine the gel is like a messy, fragile material. Transferring it to a membrane is like making a neat photocopy of the DNA pattern so you can work with it more easily. After this Nylon Mebrane baked for 2 hr at 80 degree Celsius or uv rays at 254 nm Now that the DNA is on the membrane, it’s still invisible—you can’t see where your specific DNA sequence is yet. That's where the next step comes in!

8. 4. Probing for the Specific DNA Fragment (Hybridization) After the DNA is transferred to the membrane, you need to find the specific DNA sequence you’re looking for. This is where a probe comes in. A probe is a small, single-stranded piece of DNA that is complementary to the DNA sequence you want to find. The probe is labeled with something that you can easily detect later. Common labels are radioactive isotopes (which can be detected by X-ray film) or fluorescent tags (which glow under certain lights). The membrane is exposed to the probe in a solution, allowing the probe to bind (or "hybridize") to the DNA sequence that matches it on the membrane.

9. Recap of the Process Cut the DNA into fragments with restriction enzymes. Separate the fragments by size using gel electrophoresis. Transfer the DNA from the gel to a membrane (blotting). Probe the membrane to find the specific DNA sequence you're looking for. Detect the probe to see if the DNA sequence is present.

10. "What happens if the DNA probe binds to two different people? Can two people have the same DNA?" What do you think happens if we use the wrong probe ?

11. Western Blotting: Revealing Proteins 1 Protein Extraction Break open cells to extract proteins. 2 Gel Electrophoresis Separate proteins by size. 3 Transfer to Membrane Blot proteins onto a membrane. 4 Blocking Prevent non-specific binding. 5 Antibody Probing Detect protein using antibodies. 6 Detection Visualize the protein using a reaction.

12. 1. Extracting Proteins from Cells Before doing anything, you need to extract the proteins from your biological sample (like from tissues or cells). To do this, you break open the cells using a method called lysis . This process releases all the proteins from inside the cells into a liquid solution, known as the protein lysate . Imagine you have a bunch of tiny "protein factories" (cells), and you break them open to get the raw material (proteins) out.

13. 2. Separating Proteins by Size (Gel Electrophoresis) Once you’ve extracted the proteins, you need to separate them based on their size using a method called SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). SDS is a detergent that unfolds proteins, giving them all a negative charge so they move through the gel based only on their size, not their shape. You load the protein sample into wells at the top of a polyacrylamide gel and apply an electric current . The proteins move through the gel, and just like in DNA electrophoresis, smaller proteins move faster while larger proteins move slower . This creates a pattern of proteins separated by size

14. 3. Transferring Proteins to a Membrane (Blotting) Once the proteins are separated by size in the gel, they need to be transferred to a membrane (a piece of paper-like material) so you can detect them. This is where the blotting happens. In Western blotting , the separated proteins are transferred from the gel onto a nitrocellulose or PVDF (POLYVINYLIDENE DIFLUORIDE) membrane using an electric current. This process is called electroblotting , where the electric field moves the proteins out of the gel and onto the membrane.

15. 4. Blocking the Membrane Once the proteins are transferred onto the membrane, the next step is to block the membrane to prevent non-specific binding. The membrane is coated with a blocking solution (usually made from milk proteins or BSA – Bovine Serum Albumin) to prevent random proteins or other molecules from sticking to the membrane. Blocking works like putting a protective layer on the membrane so that only your specific protein of interest will bind to the next step (the antibody), and not just any random protein.

16. 5. Probing with Primary and Secondary Antibodies This is the most crucial step, where you use antibodies to detect the specific protein you’re looking for. Primary antibody : This is the first antibody that binds to your protein of interest. It’s designed to specifically recognize and attach to your protein. Each protein has its unique structure, so the primary antibody binds only to that specific protein.For example, if you are looking for a protein called actin , the primary antibody is made to bind only to actin. Secondary antibody : The primary antibody alone is hard to detect, so a secondary antibody is used. This secondary antibody binds to the primary antibody and is tagged with a marker (like an enzyme or fluorescent dye) that allows you to see or detect the protein.

17. If Bands are formed then it indicates proteins are present in the sample

18. Proteins and Antibodies in Western Blotting Proteins Made of amino acids, perform vital functions. Antibodies Primary binds to protein, secondary carries detection label.

19. Western Blotting in Practice Detecting p53 Protein Indicates presence and function of p53 in cancer research. Visualization A band on the membrane shows the protein's size.

20. Comparing Southern and Western Blotting Feature Southern Blotting Western Blotting Detects DNA Proteins Probe Type DNA probe Antibodies Sample Type DNA from cells/tissues Proteins from cells/tissues Application Genetic mutations, DNA studies Protein expression, disease markers Detection Method Radioactive/fluorescent label Enzyme reaction or fluorescence

21. Applications of Blotting Techniques Southern Blotting Genetic disorders, forensics Western Blotting Disease diagnosis, protein studies

22. Mastering Blotting Techniques 1 Summary Essential tools in molecular biology and diagnostics. 2 Importance Mastering these techniques is key for advanced biology and medicine.