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Sunday, October 27, 2013

ELISPOT - Procedure / Protocol, Advantages, ELISA Vs ELISPOT

The Enzyme Linked Immunospot (ELISPOT)  technique was developed by Cecil Czerkinskdy in 1983. ELISPOT is used for the detection of secreted proteins, such as cytokines and growth factors. ELISPOT is primarily used in immunology research in the following areas:
  • Transplantation – prediction of infectious risk
  • Vaccine development (IFNγ)
  • Th1/Th2, T-cell regulation analysis
  • Monocyte and Dendritic cell analysis
  • Autoimmune disease
  • Cancer – tumor antigens
  • Allergy
  • Viral infection monitoring and treatment

Elispot assay

In this technique, as in ELISA 96-well plate is used but the plates will have PVDF or nitrocellulose membrane at the bottom of the well. The membranes are coated with primary antibody and the cells are grown on that wells. As the cells settle on to the membranes are secrete proteins, the protein of interest will bind to the coated primary antibody. The protein of interest can be detected using a secondary antibody.The protein will be seen as a spot of color. One spot corresponds to one cell.Using computer softwares the spots can be scanned and analyzed.

Advantages of  ELISPOT
  • Sensitive assay
  • Functional assay
  • Adaptable
Difference between ELISA and ELISPOT (ELISA Vs ELISPOT)

The enzyme-linked immunospot assay (ELISpot) resembles the enzyme-linked immunosorbent assay (ELISA). In both techniques pairs of antibodies are used: primary antibodies (catching antibodies) and secondary antibodies (detecting antibodies). Beside this, however, there are more differences than similarities. Thanks to these differences, ELISpot overcomes certain limitations of ELISA. Moreover, combining both techniques in one experiment supplies additional information, e.g. it is possible to calculate the mean production of a cytokine by single stimulated cell (e.g. pg per cell).

ELISPOT will yield information about number of cells secreting cytokine of interest (e.g. n/1 million cells)

ELISA will yield information on the Concentration of the cytokine of interest produced by all cells in the culture (e.g. pg/1 ml of supernatant)

ELISA gives you information about how much cytokine is produced and secreted at a given point of time, but no information about how many cells are secretors of the cytokine.

In ELISPOT there is possibility of detecting parallel secretion of 2 proteins by the same cell

In ELISA it is not possible to detect secretion of 2 proteins by the same cell parallely.


Cellular Technology Limited - Technical Resource
Abcam - Technical Resources
Other Internet Resources

Friday, October 25, 2013

Oligonucleotide Synthesis: Phosphoramidite Synthetic Method - Problems-Advantages

Oligonucleotide Synthesis - Phosphoramidite Synthetic Method

McBride and Caruthers in 1983, developed this method of oligonucleotide synthesis.Custom oligonucleotide synthesis begins with specification of the desired sequence in an oligonucleotide synthesis platform. Specification is composed of three crucial elements: the actual sequence that is to be made, the identification of any desired modifications, and verification of the scale at which the synthesis is to be carried out. This third element determines the choice of a column in which the synthesis will be performed. Synthesis columns 
permit a one-way flow of reagents from the synthesis platform through a precisely defined physical space containing and confining the growing oligonucleotide. The oligonucleotides are synthesized on solid supports from the 3’-end and the first monomer at this end is normally attached to a CPG(Controlled Pore Glass) or Polystyrene (PS). 

Controlled Pore Glass (CPG)

Controlled-pore glass is rigid and non-swelling with deep pores in which oligonucleotide synthesis takes place. Glass supports with 500 Å (50 nm) pores are mechanically robust and are used routinely in the synthesis of short oligonucleotides. However, synthesis yields fall off dramatically when oligonucleotides more than 40 bases in length are prepared on resins of 500 Å pore size. This is because the growing oligonucleotide blocks the pores and reduces diffusion of the reagents through the matrix. Although large-pore resins are more fragile, 1000 Å CPG resin has proved to be satisfactory for the synthesis of oligonucleotides up to 100 bases in length, and 2000 Å supports can be used for longer oligonucleotides.

Polystyrene (PS)

Highly cross-linked polystyrene beads have the advantage of good moisture exclusion properties and they allow very efficient oligonucleotide synthesis, particularly on small scale (e.g. 40 nmol).

Solid supports for conventional oligonucleotide synthesis are typically manufactured with a loading of 20-30 μmol of nucleoside per gram of resin. Oligonucleotide synthesis at higher loadings becomes less efficient owing to the steric hindrance between adjacent DNA chains attached to the resin; however, polystyrene supports with loadings of up to 350 μmol / g are used in some applications, particularly for short oligonucleotides, and enable the synthesis of large quantities of oligonucleotides.

Attached monomers are protected at the 5’-end with an acid labile and lipophilic trityl group and the A,G, C and mC monomers are protected with base labile protection groups at the nucleobase positions. Each monomer is attached through a synthetic cycle.

The Oligonucleotide Synthetic Cycle

The cycle consists of four steps:
  1. De-protection,
  2. Coupling, 
  3. Oxidation and 
  4. Capping. 
De-Protection - Oligonucleotide Synthesis:

In the classic de-protection step the trityl group attached to the 5’ carbon of the pentose sugar of the recipient nucleotide is removed by trichloroacetic acid (TCA) leaving a reactive hydroxyl group.

Coupling Step -  Oligonucleotide Synthesis:

In the coupling step, the phosphoramidite monomer is added in the presence of an activator such as a tetrazole, a weak acid that attacks the coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite intermediate. This structure then reacts with the hydroxyl group of the recipient and the 5’ to 3’ linkage is formed . The tetrazole is reconstituted and the process continues.

Oxidation Step -  Oligonucleotide Synthesis:
The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide. The traditional method of
achieving this is by treatment with iodine in water. 

Capping Step -  Oligonucleotide Synthesis:
The final step of the synthesis cycle is the capping reaction. Any remaining free 5’-hydroxyl groups are blocked at the capping step in an irreversible process. This step prevents the synthesis of oligonucleotides with missing bases. Following this step, the oligonucleotide is ready for the next monomer.

After having synthesized the full length sequence, the oligonucleotide is then released from the solid 
support using a base, such as aqueous ammonia or a mixture of ammonia and methylamine. This will also 
remove protection groups from the nucleobases. The oligonucleotide is now ready for either desalting or 
purification. For dual HPLC purification, the final trityl group is left on the oligonucleotide prior to treatment 
with ammonia. First, the oligonucleotide is purified with RP-HPLC where the retention time is to a large extent determined by the lipophilic trityl group. Following this step, the trityl group is removed and the oligonucleotide is again HPLC purified.

Advantages of Solid Phase Synthesis

Solid-phase synthesis is widely used in peptide synthesis, oligonucleotide synthesis, oligosaccharide synthesis and combinatorial chemistry. Solid-phase chemical synthesis was invented in the 1960s by Bruce Merrifield, and was of such importance that he was awarded the Nobel Prize for Chemistry in 1984.

Solid-phase synthesis is carried out on a solid support held between filters, in columns that enable all reagents and solvents to pass through freely. Solid-phase synthesis has a number of advantages over solution synthesis:
  • Large excesses of solution-phase reagents can be used to drive reactions quickly to completion
  • Impurities and excess reagents are washed away and no purification is required after each step
  • The process is amenable to automation on computer-controlled solid-phase synthesizers.
Problems and Challenges

Monitoring coupling efficiency is critical parameter to get high yield of oligo synthesis. If the coupling efficiency is 99% then, theoretical yield for a 24mer will be 89.1% full-length product (FLP) at 99.5% average coupling efficiency and 79.4% FLP at 99.0% average coupling efficiency. Even a 0.5% average coupling failure rate can be dramatic for longer oligonucleotides. A  minor increases in average coupling
failure rates will have a substantial net effect on even average length oligonucleotides. It is for this real-time monitoring of every custom synthesis reaction on every synthesis platform.

How to check the Oligo you recieved is having correct concentration???

Generally, the custom synthesized oligos which is used in PCR applications are shipped in lyophilized powder along with a datasheet. Datasheet provided along with the oligos will have all the details about the oligos (Yield, Epsilon, volume to make 100micro Molar, length, Mol. Wt, etc). For resuspending the lyophilized powder TE buffer or water can be used. the amount of water / TE buffer to be added will be mentioned on the datasheet. 

Most of the people are not aware of the fact, the oligo yield varies. To check concentration of the oligos a simple UV absorbance at 260nm will do. Once you get the OD260nm reading, using Oligocalc (an online tool) the concentration of the primers can be known. So when you recieve an oligo check the concentration after resuspension to 100uM.


IDT DNA Technical Resources
Technical Resources - Exion

Sunday, October 20, 2013

Molecular Beacons for Single Nucleotide Polymorphism Detection

Molecular beacons are short oligonucleotide hybridization probes which can report the presence of specific nucleic acid present. Molecular Beacons are mostly used in real time PCR, which can even detect single nucleotide polymorphism. This makes it very advantageous to use where detection of antibiotic resistance, allelic discrimination, diagnostic assays etc. The sequence of each molecular beacon must be customized to detect the PCR product of interest.

Molecular Beacon Basic Structure

A typical molecular beacon probe is generally >25-30 nucleotide long and will have

1. Loop
2. Stem
3. 5' Flourophore
4. 3' Quencher

Hybridization of the molecular probe to the target nucleic acid occurs if the sequence of the probe exactly matches with the target nucleic acid. Once the hybridization occurs, quencher and flourophore moves apart and results in fluorescent emission. The presence of the emission reports that the event of hybridization has occurred and hence the target nucleic acid sequence is present in the test sample.

Attached to opposite ends of the beacon are a fluorescent reporter dye and a quencher dye. When the molecular beacon is in the hairpin conformation, any fluorescence emitted by the reporter is absorbed by the quencher dye and no fluorescence is detected.
Diagram of molecular beacon: This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation.

Applications of Molecular Beacon
  • SNP detection
  • Real-time nucleic acid detection
  • Real-time PCR quantification
  • Allelic discrimination and identification
  • Multiplex PCR assays
  • Diagnostic clinical assays