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Blotting techniques (manish)

The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting for DNA, Northern blotting for RNA, and Western blotting for proteins. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe to detect specific sequences. It allows researchers to identify genes, map genomes, and study evolution and disease.

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BLOTTING TECHNIQUES Present by… CHOVATIYA MANISH M. M.SC BIOTECHNOLOGY
 Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein.
 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' Professor Sir Edwin Southern
 This method Involves separation, transfer and hybridization.  It is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples.  The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.
 Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization.  The key to this method is Hybridization.  Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.
1. The mixture of molecules is separated. 2. The molecules are immobilized on a MEMBRANE. 3. The probe is added to the membrane to bind to the molecules. 4. Any unbound probes are then removed. 5. The place where the probe is connected corresponds to the location of the immobilized target molecule.
Cellulose nitrate (nitrocellulose)  It is produced by treating cellulose with nitric acid.  Each glucose unit in the cellulose polymer is esterified with three nitrate groups, and these nitrate groups are responsible for both the negative charge of nitrocellulose at neutral pH and the unusual flammability of dry nitrocellulose.
 The major improvement has been the introduction of nylon membranes, which have three advantages over their nitrocellulose counterparts.  First,nylon membranes are less fragile than nitrocellulose sheets,the latter tending to crack if handled roughly during Southern blotting,and usually disintegrating if attempts are made to carry out more than two or three hybridization analyses with the same blot.  Nylon membranes cannot be damaged by handling and a single blot can be rehybridized up to ten times,this limit being due not to eventual breakage of the membrane but to the gradual loss of the blotted DNA during repeated hybridizations.  The second advantage of nylon membranes is that under certain conditions (a positively charged membrane and an alkaline transfer buffer) the transferred DNA becomes covalently bound to the membrane during the transfer process. This is not the case with a nitrocellulose membrane,which initially binds DNA in a semipermanent manner,immobilization occurring only when the membrane is baked at 80C.
 Transfer onto a positively charged nylon membrane can therefore reduce the possible loss of DNA that might occur by leaching through the membrane during the blotting process;  it is also quicker,the transfer time being reduced from 18 h to 2 h.  Finally,nylon membranes efficiently bind DNA fragments down to 50 bp in length,whereas nitrocellulose membranes are effective only with molecules longer than 500 bp.  Nitrocellulose has not,however,been completely superseded because it has one significant advantage compared with nylon membranes: a reduced amount of background hybridization,especially with probes that have been labelled with nonradioactive markers.
1. Digest the DNA with an appropriate restriction enzyme. 2.The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size.
The restriction fragments present in the gel are denatured with alkali TO GET ss DNA (break Hydrogen bond). To break the DNA molecules in individual bands within the gel into smaller fragments, because smaller fragments transfer more quickly than larger ones. This is achieved by soaking the gel in 0.25 molL21 HCl for 30 min,which results in a small amount of depurination – cleavage of the b-N-glycosidic bond between purine bases (adenine or guanine) and the sugar component of their nucleotides – which is followed by decomposition of the sugar structure and breakage of the polynucleotide chain.
 If a nitrocellulose membrane is being used then the alkali pretreatment is followed by neutralization of the gel by soaking in a Tris-salt buffer,this step being essential because DNA does not bind to nitrocellulose at a pH of greater than 9.0.
 Nitrocellulose membrane soaked in salt solution is placed over gel. Nucleic acid move from gel to membrane . a high concentration of salt (NaCl THAT IS the positive Na+ ions shield the negative charges on the phosphodiester backbone AND LOWERS –ve DNA = -ve membrane repelling each other  BAKING Leads to fixing of DNA strongly to the nitocellulose membrane. UV irradiation,which results in covalent attachment of DNA to a nylon membrane.
 3 METHODS OF TRANSFER : CAPILLARY  In a capillary system, rate of transfer depends on the size of the DNA, thickness of the gel, and agarose concentration.  Upward capillary transfer is slow, the architecture of the blot crushes the gel and retards diffusion of the DNA. With a high-salt buffer, it takes appr. 18 hrs to obtain acceptable transfer of a 15 kb molecule from a 5 mm thick 0.7% agarose gel; with the same gel 90% of the 1 kb molecules will be transferred in 2 hrs. This problem is partially alleviated by the depurination step, which breaks larger molecules in to fragments 1 to 2 kb in length, thereby reducing the time needed for their transfer. If the gel is thicker than 5 mm or has an agarose concentration > 1 %, it cannot be assumed that the larger fragments will have transferred to a sufficient extent after 12 hr.
The weight helps only in bringing all Capillary blotting apparatus surfaces together. High wt may break the gel.
First,the membrane is prehybridized in a solution designed to block the unused DNA binding sites on the membrane surface. If this step is omitted then the probe will bind nonspecifically to the surface of the membrane and the signal resulting from hybridization to the specific restriction fragment will be difficult if not impossible to identify. The prehybridization solution therefore contains nonbiological polymeric compounds such as polyvinylpyrrolidone and/or biological polymers such as Ficoll (a carbohydrate-based compound),bovine serum albumin or dried milk. DNA from an organism unrelated to the one whose DNA has been blotted can also be used (salmon sperm DNA is a popular choice). Prehybridization takes between 15 min and 3 h at 68C,depending on the type of membrane
 enough labeled probe DNA is added to hybridize to the target restriction fragment to produce a clear signal that can be discerned by the detection system appropriate for the label carried by the probe. The second critical factor that must be considered during the hybridization step is the specificity of the reaction. If the probe DNA has been carefully chosen then it will contain a region that is completely complementary to all or a part of the blotted restriction fragment that is being sought. If this hybridizing region in the probe is not completely complementary to the target,then it will at least have a region of strong similarity so that a stable hybrid can form. The problem is that the probe also has the potential to hybridize to any other blotted DNA fragments with which it has partial complementarity.
 Temperature is relevant because the melting temperature (Tm,the highest temperature at which the hybrid is stable) of a fully base-paired hybrid is higher than that for one in which some base pairs have not formed because the probe and target DNAs are not fully complementary. Hybridization Maximum rate occurs at 20-25°C below the Tm for DNA-DNA hybrids, 10-15°C below Tm for DNA-RNA hybrids  Formation of the desired hybrid,and destabilization of nonspecific hybrids,can therefore be achieved by utilizing an appropriate combination of buffer composition and hybridization temperature.
 The probe hybridizes to the complementary DNA restriction fragment. Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized.
 Southern blots are used in gene discovery , mapping, evolution and development studies, diagnostics and forensics (It is used for DNA fingerprinting, preparation of RFLP maps)  identification of the transferred genes in transgenic individuals, etc.
 Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples.  Southern blot is used to detect the presence of a particular bit of DNA in a sample  analyze the genetic patterns which appear in a person's DNA.
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 first is to determine which tissues express a particular gene, and this can give some indication of the physiological function of the encoded protein.  The second principal reason for measuring an mRNA is to determine the factors which regulate the expression of a given gene, be they nutritional, hormonal, or environmental.
 Northern blotting.  A second method is the RNase protection assay, which is generally considered to offer improved sensitivity.  The third method utilizes the reverse transcriptase polymerase chain reaction; this provides considerable increases in sensitivity over Northern blotting and the RNase protection assay, and may be useful for measuring very low levels (a few copies) of an mRNA in a tissue.
 Then, in a single step reaction, the nucleases are inactivated and the remaining probe:target hybrids are precipitated. These products are separated on a denaturing polyacrylamide gel and are visualized by autoradiography.
1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) 1. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail
2.Sample’s are loaded on gel and the RNA samples are separated according to their size on an agarose gel .  The resulting gel following after the electrophoresis run.
3. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement. Nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them.
 Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.
4. The membrane is placed in a dish containing hybridization buffer with a labeled probe.  Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 5. The membrane is washed to remove unbound probe.
6. The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film.  Now the expression patterns of the sequence of interest in the different samples can be compared.
 The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure.  Polyacrylamide gel electrophoeresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs
 A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts)  Detection of mRNA transcript size  Study RNA degradation  Study RNA splicing  Study RNA half-life  Often used to confirm and check transgenic / knockout mice (animals)
 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.  Detects proteins and estimates their molecular weight.  Used to detect changes in protein expression.
 Tissue preparation  Samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, virus or environmental samples can be the source of protein and thus western blotting is not restricted to cellular studies only.  Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing and degradation.  A combination of biochemical and mechanical techniques – comprising various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.
1. After the samples have been prepared, they are separated by size using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) . 1. Since the samples have been denatured in gel loading buffer containing SDS detergent, the protein is uniformly negatively charged and will now migrate in an electric field through the gel and towards the positive electrode .
 SDS-PAGE ( sodium dodecylsulphate-polyacrylamide gel electrophoresis)  The purpose of this method is to separate proteins according to their size, and no other physical feature.
 Stacking Gel
 Transfer of the proteins fractionated by SDS-PAGE to a solid support membrane (Western blotting) can be accomplished by electroblotting
 In this procedure, a sandwich of gel and solid support membrane (Nitrocellulose or PVDF) is compressed in a cassette and immersed in buffer between two parallel electrodes.  A current is passed at right angles to the gel, which causes the separated proteins to electrophorese out of the gel and onto the solid support membrane
 The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps.  A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal to noise ratio. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics. For true optimization, empirical testing of blocking buffers is essential.
 The balance of SDS in the transfer buffer, protein size, and gel percentage are the main factors that affect transfer efficiency.  About the current and transfer time for western blot, it's critical to choose the appropriate current and transfer time for a successful western blotting.  Too low current or/and transfer time will lead to incomplete transfer; if the current or/and transfer time is too high, on the contrary, the proteins may migrate through the membrane too fast without being absorbed.
 One of the critical features of any successful Western blot is the highly  specific interaction between an antibody and an antigen. The antigen,  usually a protein or peptide, is the target of the antibody. The precise point  of interaction is between a small region of the antigen, an epitope,  and the recognition sites found on the arms of the antibody molecule.
1.The confirmatory HIV test 2.Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE( 3.Some forms of Lyme disease testing employ Western blotting .
Blotting techniques (manish)