Science Class Presentation in Pink Blue Flat Graphic Style_20240922_102006_0000.pptx

ied because they allow scientists to detect, identify, and characterize specific DNA, RNA, and proteins within a complex biological sample, providing crucial insights into genetics, gene expression, and protein function. By transferring separated macromolecules from a gel to a solid membrane and using specific probes or antibodies, researchers can study gene expression, diagnose diseases, and identify infectious agents or genetic mutations.

Key applications of blotting techniques:
Southern Blotting (DNA):
Used to detect specific DNA sequences and genetic mutations, such as those linked to diseases like sickle cell anemia.
Northern Blotting (RNA):
Helps researchers understand gene expression and regulation by detecting specific RNA transcripts.
Western Blotting (Protein):
Used to identify specific proteins and is crucial in diagnosing diseases like HIV and Lyme disease, detecting cancer biomarkers, and analyzing protein function.
Dot Blotting:
A simpler method for identifying specific DNA or RNA sequences.
Why they are studied and used:
Research:
They are fundamental tools for advancing our understanding of molecular biology, genetics, and disease mechanisms.
Diagnostics:
Blotting techniques play a vital role in clinical settings, from identifying infectious agents to diagnosing genetic disorders and certain cancers.
Specificity:
The use of probes (for nucleic acids) or antibodies (for proteins) ensures high specificity in detecting target molecules, even in a mixture of thousands of molecules.
Immobilization:
Transferring the separated molecules from a gel to a membrane immobilizes them, making them easier to probe, detect, and analyze.

1. By:-Narendra Shende M.Sc.1 st year 1 st Sem L I T University Nagpur Guided by :- Tumane Sir Presentation 2024 𝐁𝐥𝐨𝐭𝐭𝐢𝐧𝐠 𝐭𝐞𝐜𝐡𝐧𝐢𝐪𝐮𝐞𝐬

2. Blotting techniques are a set of laboratory procedures used to transfer molecules, such as DNA, RNA, or proteins, from a gel to a membrane. This transfer allows for easier detection and analysis of these molecules Introduction

3. Types of blotting techniques 1) Southern blotting (to detect DNA) 2) Northern blotting (to detect RNA) 3) Western blotting (to detect protein)

4. SOUTHERN BLOTTING Professor Sir Edwin Southern, Professor of Biochemistry and Fellow of Trinity developed this method in 1975. Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting'

5. Blotting techniques are based on the principle of molecular hybridization. This refers to the ability of complementary nucleic acid sequences (DNA or RNA) to bind to each other through base pairing (A- T, G- C). Principle

6. STEPS Restriction enzyme are needed to cleave the DNA sample into fragments prior to electrophoresis Prior to blotting, the separated double- stranded DNA are denatured with alkaline solution → single- stranded DNAfragmentsBlotting method: capillary transfer of DNA from an electrophoresis gel to a filter Uses labeled oligonucleotide probes that are complementary to the target DNA sequence Incubation allows hybridization of the probe with the target DNA

7. DNA in a Application identify a specific DNA sample identification of a single gene in a pool of DNA fragments. gene mapping. analysis of genetic patterns of DNA. detection of specific DNA sequences in a genome. study of gene deletions, duplications, and mutations that cause various diseases.

8. Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting The principle of Northern blotting is based on the ability of complementary RNA and DNA strands to form stable complexes through base pairing.

9. Steps 1.Separating RNA RNA is isolated from a biological sample and separated by size using gel electrophoresis. Transferring RNA The separated RNA is transferred from the gel to a solid membrane. Hybridizing A labeled probe is added to the membrane to hybridize with the RNA of interest. Detecting The hybridized complex is detected using a radioactive, fluorescent, or chemical tag.

10. Application study RNA and can be used to: Determine the abundance and size of RNA transcripts, Detect transcript variants of genes, Evaluate gene expression levels, Detect viral microRNAs, and Diagnose diseases like Crohn's disease.

11. Western blotting Western blotting (1981) is an Immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules. The SDS PAGE technique is a prerequisite for Western blotting. Principle Equal loading of proteins, separation of proteins by molecular weight, electrophoretic transfer to a suitable membrane, and probing of antibodies.

12. Steps . Protein separation A mixture of proteins is separated by gel electrophoresis based on their molecular weight and electrical properties. Protein transfer The separated proteins are transferred to a membrane, such as nitrocellulose or polyvinylidene difluoride (PVDF). Antibody probing The membrane is incubated with antibodies that are specific to the protein of interest. The unbound antibodies are washed off, leaving only the bound antibodies. Detection The bound antibodies are detected by developing the film. The thickness of the band corresponds to the amount of protein present.

13. Application Western blotting has many applications in biotech and medical labs for research and diagnostic purposes. It's used to study proteins, quantify their concentration in a specific sample, and help in the identification of diseases, such as Lyme disease, HIV infection, bovine spongiform encephalopathy (BSE), hepatitis C infection, syphilis, inflammatory muscle conditions such as myositis, and certain autoimmune disorders (e.g., paraneoplastic disease)

14. THANK YOU! For Your Attention With Mr.Narendra