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Blotting techniques includes southren,northern,western and dot blotting

This document describes various blotting techniques used to detect specific DNA or RNA sequences, including Southern blotting, Northern blotting, Western blotting, and dot blotting. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane and probing for specific sequences. Northern blotting is similar but uses RNA. Western blotting detects specific proteins. Dot blotting detects sequences in non-fractionated samples by directly applying samples to a membrane. These techniques allow for detection and analysis of genetic material.

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BLOTTING TECHNIQUES BINCY MARIAM YESUDAS MSC BOTANY
INTRODUCTION  Gel electrophoresis techniques enables us to separate fragments of DNA in a mixture and by staining we can visualise and detect them.  Use of specific labelled probes enables in detection and identifications of fragments of DNA by hybridization procedure.  To facilitate this hybridization , the bands are often transferred to a nitrocellulose membrane. This technique resembles blotting.  For such blotting, DNA has to be single stranded form. This can be achieved by denaturation of DNA.
BLOTTING TECHNIQUES-DIFFERENT TYPES 1.SOUTHERN BLOTTING 2.NORTHERN BLOTTING 3.WESTERN BLOTTING 4.DOT BLOT TECHNIQUE
SOUTHERN BLOTTING  Southern blotting is the first blotting technique which made analysis and recording easy.  In Southern blotting ,a DNA fragment containing a specific sequence can be identified by electrophoresis , transferring them into nitrocellulose & hybridizing with a 32p labelled single stranded DNA probe complementary to the sequence.  The fragment containing the sequence then visualized by audioradiography  It was first developed by E.M.Southern in 1975
PROCEDURE  For Southern blotting,DNA sample is first digested with a restriction enzyme and digested sample is electrophoresed.  The DNA bands in the gel are denatured into single strands with the help of an alkali solution.  Subsequently the gel is laid on top of a buffer saturated filter paper placed on a solid support(eg.glass plate),with its two edges immersed in the buffer.  A sheet of nitrocellulose filter membrane is placed on top of the gel and a stack of many papers on top of this membrane.
 Capillary action pulls the buffer from the bottom filter through the gel, to the transfer medium and up through the paper towel stack.  While passing through the gel, the buffer carries with its single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels.  The nitro cellulose membrane with single stranded DNA bands blotted on to it, is baked at 80c for 2-3 hours to fix the DNA permanently on the membrane.  DNA fragments on membrane can then be probed for sequence of interest by hybridization between them.  Membrane is washed to remove the unbound DNA.  X-ray film is then exposed to the membrane to get autoradiographs.
SOUTHERN BLOTTING
APPLICATIONS  Southern blotting is extremely sensitive and can be applied to mapping restriction sites around a single copy gene sequence in a complex genome such as that of man.  When minisatellite probe is used the technique can be also used for forensic purpose for identification of minute amount of DNA.  The use of southern blot technique has also been done for analyzing the role of DNA methylation in gene expression.
 Blotting analysis useful in detection of mutated genes in genetic disorders.  Restriction analysis of genes studied with southern blot helps in this respect.  RFLP mapping.  DNA finger printing.
NORTHERN BLOTTING  The Northern blot procedure essentially identical to that of southern blotting except that here RNAs are separated by gel electrophoresis.  Initially southern blotting could not be used directly to blot transfer RNA from gel to nitrocellulose membrane because RNA did not bind to cellulose nitrate.  Alwine,Kemp and Stark(1979)developed a procedure for blot transfer of RNA.  He used a chemically reactive paper(diazotization of amino benzyl oxymethyl paper which is prepared from Whatsman 540 papers).
PROCEDURE  RNA species are separated on the basis of size by electrophoresis through agarose gel containing formaldehyde or glyoxal and dimethyl sulfoxide.  The separated RNA bands are then blotted on chemically reactive filter paper.  RNA species after blotting are hybridized to radiolabelled DNA probe.  Auto radiography is carried onto locate RNA bands that are complementary to the probe.
NORTHERN BLOTTING
APPLICATIONS  Used for detection and quantitative estimation of hybridized mRNA.  Study RNA degradation  Study RNA half life  Study RNA splicing  It is useful in the studies of gene expression.
WESTERN BLOTTING  This technique is used to detect the proteins of a particular specificity.  When a transferred gene expresses in transformed cells,  The translated product in the form of proteins can be identified by this technique.
PROCEDURE  First we isolate the protein or extract the protein.  The extracted proteins are subjected to PAGE(Poly Acrylamide Gel Electrophoresis) and are then transferred onto nitro cellulose to which they bind.  Then radiolabelled specific antibody is added on such membrane and it binds only to specific complementary protein.  The antibody is labelled with 125 I and the signal is detected again with autoradiography.  If radio active label is not used, bound antibody may be detected by a second antibody tagged with an enzyme.
APPLICATIONS  The confirmatory HIV test employs a western blot to detect anti- HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.  A western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').  Western blot can also be used as a confirmatory test for Hepatitis B infection.
DOT BLOT TECHNIQUE  This technique is used to detect the presence of a given sequence of DNA/RNA in the non- fractionated(not subjected to electrophoresis) DNA sample.  DNA from many samples can be tested in a single test.  A Dot blot (or Slot blot) is a technique used to detect biomolecules.  It represents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated
 Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot.  Then is spotted through circular templates directly onto the membrane or paper substrate. .  Then followed by detection by either nucleotide probes (for a northern blot and Southern blot) or antibodies (for a western blot).
PROCEDURE  Sample DNA/RNA from different individuals are transferred to a nitrocellulose filter paper in the form of dots. Several samples are blotted on to a single filter paper.  The DNA is first denatured and then the filter is baked at 80c to fix DNA firmly on to the filter.  The filter is treated with the appropriate radio active single stranded probe under conditions favouring hybridization.The filter is washed to remove the unhybridized probes.  The dots with hybridized probes are detected by autoradiography intensity of the dots corresponds fairly with the extent to which DNA/RNA is represented in the sample.
 The dot blot test can be used to detect the Chlamydia trachomatis infection and other sexually transmitted diseases.  Dot blot is used to detect Antidiacyltrehalose Antibodies in Tuberculosis patients and typhoid Fever
Blotting techniques includes southren,northern,western and dot blotting