Dr. Aamir Ali PhD (Biotechnology), MPhil,Health Biotechnology Division, NIBGE

Selection and Identification of Recombinant clones

After transformation, we challenge the bacteria with anantibiotic (such as ampicillin). If the E. coli have taken up andexpressed an ampicillin resistance gene on a plasmid, they willlive - otherwise they will die. This process is called selection,because we are selecting which of the bacteria can survive.

Selection of Recombinant Bacteria

Selection is a process designed to facilitate the identification ofrecombinant bacteria while preventing the growth of non-transformed bacteria

Antibiotic selection

Blue-white selection

Selection

Antibiotic selection

for selection of transformants

Blue-white selection

for selection of recombinant clones

Non-functioning (insertional deactivation) of lacZ geneshows that the gene of interest has properly been insertedinto the MCS.

Therefore the recombinant clones (white colonies) can befurther analyzed for production of the recombinantprotein/product.

Nucleic Acid Hybridization

Southern/Northern Blotting

1. Identification of recombinant clones

2. Direct identification of restricted DNA

Southern Hybridization

Hybridization probing is usually the preferred method for identification of aparticular recombinant from a clone library. The technique is easy to performand, with modifications introduced in recent years, can be used to check upto 10,000 recombinants per experiment, allowing large genomic libraries tobe screened in a reasonably short time.

Nevertheless, the requirement for a probe that is at least partlycomplementary to the desired gene sometimes makes it impossible to usehybridization in clone identification. On these occasions a different strategyis needed.

The main alternative to hybridization probing is immunological screening.The distinction is that, whereas with hybridization probing the cloned DNAfragment is itself directly identified, an immunological method detects theprotein coded by the cloned gene. Immunological techniques thereforepresuppose that the cloned gene is being expressed, so that the protein isbeing made, and that this protein is not normally present in the host cells.

There are occasions when it is necessary to usehybridization probing to identify which of a series ofrestriction fragments contains a gene of interest.

The method called Southern hybridization enables theindividual restriction fragment containing a particulargene to be identified.

Southern Hybridization

This method was developed by a molecular biologist E.M Southern (1975)for analyzing the related genes in a DNA restriction fragment.

In Southern Hybridization, a sample of DNA containing fragments ofdifferent sizes is subjected to electrophoresis using either poly-acrylamideor agarose gel.

The DNA sample may either be subjected to mechanical shearing or torestriction digestion in order to generate the fragments.

Agarose gel is useful to separating DNA fragments of few hundred to 20kb insize while polyacrylamide is preferred for smaller fragments.

In many situations, it is critical to detect and identify DNA fragments in asample that are complementary to a given DNA sequence. This is achievedby Southern Hybridization.

1. The restriction fragments of DNA present in agarose gel are denatured intosingle stranded form by alkali treatment.

2. They are then transferred onto a nitrocellulose filter membrane. This is doneby placing the gel on the top of the buffer saturated filter paper, then laying thenitrocellulose filter membrane on the top of the gel, and finally placing some dryfilter papers on the top of the membrane. The dry filter paper draws the bufferthrough the gel.

The buffer moves due to capillary action,from the bottom filter paper through thegel carrying with the denatured DNApresent in the gel, the DNA becomestrapped in the nitrocellulose membraneas buffer phases through it. This processis known as blotting and takes severalhours to complete.

The relative position of the bands on themembrane remain the same as that in thegel.

Procedure:

3. The nitrocellulose membrane is now removed from the blotting stack and theDNA is permanently immobilized on the membrane by baking it at 80 0C invacuum.

4. ssDNA has a high affinity for nitrocellulose filter paper. Therefore , the bakedmembrane is treated with a solution containing 0.2% each of ficoll (an artificialpolymer of sucrose), polyvinyl pyrolidone and BSA. This treatment preventsnon specific binding of the radioactive probe probably by attachingmacromolecules to all the free binding sites on the membrane. It is known aspretreatment.

5. The pretreated membrane is placed in a solution of radioactive, ssDNAor an oligonucleotide called probe which hybridizes with thecomplementary DNA on the membrane to the grater extent with alow nonspecific binding on the membrane, this is known ashybridization reaction.

6. After hybridization reaction the membrane is washed to remove theunbound probes.

7. The membrane is now placed in close contact with an X-ray film andincubated for desired period to allow images. The film is thendeveloped to reveal distinct band indicating positions in the gel of theDNA fragments that are complementary to the radioactive probe usedin the study.

Southern blots performed with restriction enzyme-digested genomic DNA maybe used to determine the number of sequences (e.g., gene copies) in a genome.A probe that hybridizes only to a single DNA segment that has not been cut bythe restriction enzyme will produce a single band on a Southern blot, whereasmultiple bands will likely be observed when the probe hybridizes to severalhighly similar sequences.

Modification of the hybridization conditions (for example, increasing thehybridization temperature or decreasing salt concentration) may be used toincrease specificity and decrease hybridization of the probe to sequences thatare less than 100% similar.

The Southern Blotting Technique is extremely sensitive. It can be used to mapthe restriction sites and DNA fingerprinting.

It is used for detection and identification of the transferred genes in transgenicindividuals.

This approach is useful when few DNA fragments with considerable lengths areto be separated and studied.

Applications

The northern blot is a technique used in molecular biology research to studygene expression by detection of RNA (or isolated mRNA) in a sample.

With northern blotting it is possible to observe cellular control over structureand function by determining the particular gene expression levels duringdifferentiation, morphogenesis, as well as abnormal or diseased conditions.

Northern blotting involves the use of electrophoresis to separate RNAsamples by size and detection with a hybridization probe complementary topart of or the entire target sequence.

The term 'northern blot' actually refers specifically to the capillary transfer ofRNA from the electrophoresis gel to the blotting membrane. However, theentire process is commonly referred to as northern blotting.

Northern Blotting:

A general blotting procedure starts with extraction of total RNA from ahomogenized tissue sample or from cells. Eukaryotic mRNA can then beisolated through the use of oligo (dT) cellulose chromatography toisolate only those RNAs with a poly(A) tail.

RNA samples are then separated by gel electrophoresis. Since the gelsare fragile and the probes are unable to enter the matrix, the RNAsamples, now separated by size, are transferred to a nylon membranethrough a capillary or vacuum blotting system.

A nylon membrane with a positive charge is the most effective for use innorthern blotting since the negatively charged nucleic acids have a highaffinity for them. The transfer buffer used for the blotting usuallycontains formamide because it lowers the annealing temperature of theprobe-RNA interaction, thus preventing RNA degradation by hightemperatures.

Procedure:

Once the RNA has been transferred to the membrane, it isimmobilized through covalent linkage to the membrane by UV light orheat. After a probe has been labeled, it is hybridized to the RNA on themembrane. Experimental conditions that can affect the efficiency andspecificity of hybridization include ionic strength, duplex length,mismatched base pairs, and base composition.

The membrane is washed to ensure that the probe has boundspecifically and to avoid background signals from arising. The hybridsignals are then detected by X-ray film.

Northern Blotting:

Northern blotting allows one to observe a particular gene's expressionpattern between tissues, organs, developmental stages, environmentalstress levels, pathogen infection, and over the course of treatment.

The technique has been used to show over-expression of oncogenes anddown-regulation of tumor-suppressor genes in cancerous cells whencompared to 'normal' tissue, as well as the gene expression in the rejectionof transplanted organs.

The expression patterns obtained under given conditions can provide insightinto the function of that gene.

The variance in size of a gene product can also indicate deletions or errors intranscript processing, by altering the probe target used along the knownsequence it is possible to determine which region of the RNA is missing.

Applications of Northern Blotting:

BlotBase is an online database publishing northern blots.BlotBase has over 700 published northern blots of humanand mouse samples, in over 650 genes across more than 25different tissue types. Northern blots can be searched by ablot ID, paper reference, gene identifier, or by tissue.

Dr. Aamir Ali PhD (Biotechnology), MPhil,Health Biotechnology Division, NIBGE

Protein blotting (Western Blotting)

Western Blotting

The method originated in the laboratory of George Stark at Stanford.

Protein immunoblot (Western Blotting) is an analytical technique, used todetect specific proteins in the given sample (tissue homogenate/extract).

It uses gel electrophoresis to separate native proteins by 3-D structure ordenatured proteins by the length of the polypeptide.

The proteins are then transferred to a membrane, where they are probed(detected) using antibodies (commercially available) specific to the targetprotein.

Steps in a Western blot

Tissue Preparation

Gel Electrophoresis

Transfer

Blocking

Detection

Tissue preparation Samples can not be taken from whole tissue or from cell culture.

Solid tissues are first broken down mechanically using ablender/homogenizer or by sonication.

Different detergents, salts, and buffers may be employed toencourage lysis of cells and to solubilize proteins. Protease andphosphatase inhibitors are often added to prevent the digestionof the sample by its own enzymes.

Tissue preparation is often done at cold temperatures to avoidprotein denaturing and degradation.

A combination of biochemical and mechanical techniques comprising various types of filtration and centrifugation can beused to separate different cell compartments and organelles.

The proteins of the sample are separated using gel electrophoresis and themost common type of gel electrophoresis employs polyacrylamide gels andbuffers loaded with sodium dodecyl sulfate (SDS).

SDS-PAGE maintains polypeptides in a denatured state once they have beentreated with strong reducing agents to remove secondary and tertiarystructure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) andthus allows separation of proteins by their molecular weight.

Sampled proteins become covered in the negatively charged SDS and moveto the positively charged electrode. Smaller proteins migrate faster throughthis mesh and the proteins are thus separated according to size (usuallymeasured in kilodaltons, kDa).

The concentration of acrylamide determines the resolution of the gel - thegreater the acrylamide concentration the better the resolution of lowermolecular weight proteins. The lower the acrylamide concentration thebetter the resolution of higher molecular weight proteins.

Gel Electrophoresis

Transfer

The proteins are moved from within the gel onto a membrane made ofnitrocellulose or polyvinylidene difluoride (PVDF). The proteins move whilemaintaining the organization they had within the gel.

Transfer involves placing a membrane on top of the gel, and a stack of filterpapers on top of that. The entire stack is placed in a buffer solution which movesup the paper by capillary action, bringing the proteins with it.

Instead of this, the preferred method for transferring the proteins is calledelectroblotting which uses an electric current to pull proteins from the gel intothe membrane.

Protein binding is based upon hydrophobic interactions, as well as chargedinteractions between the membrane and protein. The nitrocellulose membranesare cheaper than PVDF, but are far more fragile and do not stand up well torepeated probings.

Blocking

Since the membrane has been chosen for its ability to bind protein and asboth antibodies and the target are proteins, steps must be taken to preventinteractions between the membrane and the antibody used for detection ofthe target protein.

Blocking of non-specific binding is achieved by placing the membrane in adilute solution of protein - typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered Saline (TBS), with a minutepercentage of detergent such as Tween 20 or Triton X-100.

The protein in the dilute solution attaches to the membrane in all placeswhere the target proteins have not attached. Thus, when the antibody isadded, there is no room on the membrane for it to attach other than on thebinding sites of the specific target protein. This reduces "noise" in the finalproduct of the western blot, leading to clearer results, and eliminates falsepositives.

DetectionDuring the detection process the membrane is "probed" for the protein ofinterest with a modified antibody which is linked to a reporter enzyme;when exposed to an appropriate substrate this enzyme drives acolourimetric reaction and produces a color.

For a variety of reasons, this traditionally takes place in a two-step process,although there are now one-step detection methods available for certainapplications.

Primary antibodies are generated when a host species or immune cellculture is exposed to the protein of interest or its part.

A dilute solution of primary antibody (generally between 0.5 and 5micrograms/mL) is incubated with the membrane under gentle agitation for30 minutes to overnight.

After rinsing the membrane to remove unbound primary antibody, themembrane is exposed to another antibody, directed at a species-specificportion of the primary antibody. For example, anti-mouse secondary willbind to almost any mouse-sourced primary antibody. This is known as asecondary antibody, and due to its targeting properties, tends to be referredto as "anti-mouse," "anti-goat," etc.

The secondary antibody is usually linked to biotin or to a reporter enzymesuch as alkaline phosphatase or horseradish peroxidase. Therefore, onaddition of particular substrate a colored product is formed and detected.

Two-step process

Membrane

Protein of interest

Primary antibody(raised in mice)

Secondary Antibody linked with enzyme (Goat anti-mouse Ab)

Two-step process

Substrate

Colored Product

Immuno-diffusion assayImmuno-diffusion assay was designed and performed on 1% agarose by

Ouchterlony and Nilson.

Steps involved

1. Preparation of plates

2. Inoculation of plates

3. Staining and incubation of plates

4. De-staining of plates

Preparation of plates

Washed, dried glass plates (pre-coated with 0.1% agarose in water) areplaced on clean, leveled surface.

A clear colorless solution of agarose (1%) in saline is prepared by heatingand stirring.

An approximate amount (3ml or 9 ml) of agarose solution is delivered,slowly and constantly to slides.

Let the slides sit at room temperature for 15 minutes to solidify theagarose.

Placed and stored the slides in moist environment in refrigerator till furtheruse.

Inoculation of plates

The gel is cut in desired size and pattern of wells by vacuum cuttingapparatus.

Each well is filled with appropriate amount (5~15 ml) of reactant neededaccording the pattern of experiment.

Incubate the plate overnight in moist environment in refrigerator.

Wash the plates in normal saline for one hour with one saline change after 30 minutes.

Remove the plates from saline and put on paper towels

Cover the gel with wrinkle free filter paper and few layers of towels.

Press the gel (under heavy books) for 2 hours with one paper towels change.

Air dry the plates at room temperature.

Staining and De-staining of plates

Soak the plates in staining solution (Coomassie blue dye (0.2% wt/vol) insolution of 45% ethanol, 45% pyrogen free water and 10% glacial aceticacid) for 5 minutes.

Remove the staining solution and immediately put the plate in de-stainingsolution (45% ethanol, 45% pyrogen free water and 10% glacial acetic acid)till the background became clear.

Air dried the plates at room temperature and stored.

DT

Anti-DT

Immuno diffusion assay of different fractions from the column

Immuno diffusion assay

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