BLOTTING TECHNIQUES

BLOTTING TECHNIQUESBY SHEELA09HA013

What is blotting techniques?It is a tool that is widely used for identification of desired DNA or RNA fragments from thousands of molecules.

Blotting refers to the process of sample nucleic acids on solid support(nitrocellulose & nylon membrane).TYPES OF BLOTTING TECHNIQUES:SOUTHERN BLOTTING (for DNA)

NORTHERN BLOTTING (for RNA)

WESTERN BLOTTING (PROTEINS)

DOT BLOTTING (DNA/RNA)OUTLINE OF NUCLEIC ACID OF BLOTTING TECHNIQUE IMMOBILIZATION OF NUCLEIC ACID SOUTHERN BLOT(DNA) NORTHERN BLOT(RNA) WESTERN BLOT(PROTEINS) PREHYBRIDIZATION HYBRIDIZATION STRINGENCY WASHES DETECTIONSOUTHERN BLOTTINGIt was named after scientist Southern who developed it in 1975.

Southern blotting is mainly meant for DNA.

Genomic DNADNA fragments

The genomic DNA is isolated from cells/tissues

Digestion of the DNA using one or more restriction enzymes.

The fragments are loaded into the well of agarose or polyacrylamide gel it is subjected to electrophoresis.

DNA being negatively charged it migrate towards anode & we know that smaller fragments move faster.The separated DNA molecules are denatured by exposure to a mild alkali , then it is transferred to nitrocelluose or nylon membrane which creates exact replica of DNA fragments on the gel

The DNA is annealed to nitrocellulose by exposing to heat of (80 C)

The nitrocellulose is exposed to labeled cDNA probes.

These probes hybridize with complementary DNA .After thorough washing the paper is exposed to X ray film to develop autoradiograph.

This shows the specific bands corresponding to the DNA fragments recognized by cDNA probe.

NORTHERN BLOTTING:

It is similar to southern blotting expect that RNA is blotted through this method this was named as jargons (since it was named after scientist Southern).

Instead of nitrocellulose membrane chemically reactive paper prepared by diazotization of aminobenzyloxymethyl to create diazobenzyloxymethyl (DBM) paper. The RNA covalently bind to DBM paper.

But they modified nitrocellulose membrane, at present it is been used for northern blotting.

The same procedure follows where the blot is transferred RNA molecules hybridize with DNA probe which can be detected.

Northern blotting is a technique for determining the number of genes present in given DNA, each DNA gives rise to two or more RNA transcripts, but the drawback is the presence of exons an introns.

DOT BLOTTINGModification of southern and northern blotting where the nucleic acids(DNA or RNA) are directly spotted onto filters and not subjected to electrophoresis the hybridization technique is similar.

This technique is particularly useful in obtaining quantitative data for the evaluation of gene expression.

WESTERN BLOTTINGThis involves identification of proteins, useful to understand the nucleic acid functions. This is also called as immunoblottings

This involves the transfer of electrophoresed protein bands from polyacrylamide gel to nylon or nitrocellulose membrane. These proteins can be detected by specific protein ligand interactions. Antibodies or lectins are commonly used for this purpose. Applications of Blotting Techniques -

It is method of gene analysis.

Important for the confirmation of DNA cloning results

Forensically applied to detect minute quantities of DNA (parenthood, thieves etc.)

DNA pieces from one species (eg; human) can be used to detect DNA molecules from related species ( e.g: chimpanze) this is referred to as Zoo- blotting.

Southern blotting technique is widely used to find specific nucleic acid sequence present in different animals including man. For example if we want to know whether there is a gene like insulin in sea anemone, then DNA of sea anemone is mobilized on membrane and blotted by using insulin probes against it.

Northern blotting technique is widely used to find gene expression and regulation of specific genes. For example if we find human insulin like gene in oyster, then by isolating and immobilizing RNA and blotting it with insulin probe we call tell whether the gene is expressing or not.

By using blotting technique we can identify infectious agents present in the sample.

We can identify inherited disease.

It can be applied to mapping restriction sites in single copy gene.

Highly useful for the determination of restriction fragment length polymorphism (RFLP) associated with pathological conditions.

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