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Friday, December 6, 2013

Real Time PCR (qPCR) - Fluorescent Primer Probe Based Chemistries

Fluorescent Primer Probe based chemistries are the core of real time pcr (qPCR). The most common fluorescent based chemistries are Taqman Probes and Molecular Beacons. Other fluorescent primer probe based chemistries includes  Eclipse probes and Amplifluor, Scorpions, LUX, and BD QZyme primers.

A Typical Real Time PCR Amplification Plot (Ct Vs Fluorescence)

All the above mentioned Primer - Probe based detection chemistries take advantage of Fluorescence Resonance Energy Transfer (FRET), or some other form of fluorescence quenching,which will  ensure that the specific fluorescence is detected only in the presence of amplified product.

TaqMan Probe Chemistry

TaqMan = Taq Polymerase + PacMan




TaqMan assay includes an oligonucleotide probe called the TaqMan Probe, containing a fluorescent reporter dye at the 5' end and quencher at the 3' end. This oligonucleotide is developed in such a way that it will bind to downstream of the primer binding site in the target DNA molecule.

TaqMan assay utilizes the exonuclease activity of taq DNA polymerase, during the elongation step in the PCR, taq DNA polymerase displaces the bound reporter dye.

Due to the 5'-3' exonuclease activity of Taq DNA Polymerase, TaqMan probe will be removed / cleaved from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.

when the reporter and quencher are inn close proximity, the fluorescence emitted by the reporter dye will be readily quenched by the quencher, when the reporter is cleaved by the taq DNA polymerase, reporter moves away from the quencher thereby no quenching occurs, resulting in the emission of fluorescent signal.

TaqMan Assay Vs SYBR Green Assay

Molecular Beacons

In molecular beacon assay other than the two specific primers, a flourescently labelled oligonucleotide probe called the "molecular beacon", a dye-labelled oligonucleotide which forms a hairpin structure with stem and a loop.

Structure of Molecular Beacon


molecular beacon probe



Molecular Beacon is usually  25-40 nucleotides long. molecular beacon has a flourescently labelled reporter at the 5' end and quencher at the 3' end. Typically a Molecular Beacon has:

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

The Loop of molecular beacon is designed in such a way that it will hybridize to 15-30 nucleotide section of the target nucleic acid. As from the image above we can make out that the stem region is a set of complementary sequence which forms a stem like structure which keep the reporter and quencher close by. As a result of this hair pair pin form no fluorescence is detected from the reported because of the presence of quencher near to it. During the annealing step of the pcr reaction, molecular beacon binds to the target nucleic acid opening up the hairpin structure and as result the reporter and quencher moves father apart, so fluorescence is detected from the reporter. The amount of fluorescence emitted by the reporteron the molecular beacon is directly proportional to the amount of target in the reaction.

Difference between Molecular Beacon Assay and Taqman Assay

The main difference is that in molecular beacon assay Taq Polymerase used will lack 5'-3' exonuclease activity so here in molecular beacon assay molecular beacons are not destroyed. But in Taqman assay taq DNA polymerase with 5'-3' exonuclease activity is used which will cleave the probe.

Advantages of Molecular Beacons
  1. Highly Specific compared to other Assays.
  2. Multiplexing Possible
  3. Allelic discrimination and identification
  4. Used for Single Nucleotide Polymorphisms.
Disadvantages of Molecular Beacons
  1. Difficult to design
  2. Unintended fluorescence can be produced if the hairpin opens into non-hairpin confirmation.
Hybridization Probe Assay

In Hybridization probe assay,In addition to two sequence specific primers, two sequence specific oligonucleotide probes are used.


There are two oligonucleotide probes, Probe 1 having donor dye at 3' end and the Probe 2 carries a acceptor dye at the 5' end. The dyes of donor and acceptor probes are selected in such a way that emission spectrum of the donor dye overlaps the excitation spectrum of the acceptor dye, where as the emission spectrum of the donor dye is separated from the emission spectrum of the acceptor dye. Excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. During the annealing step of PCR, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the fluorescent molecules into proximity, allowing fluorescence resonance energy transfer from donor to acceptor. The increasing amount of acceptor fluorescence is proportional to the amount of amplicon present.

Minor Groove Binder (MGB) Eclipse Probe

Eclipse Probe Assay will have two primers and a sequence specific oligonucleotide probe. The probe will bind to the specific target sequence, the specialty of MGB Eclipse Probe is that it has a 5' Quencher and a 3' Reporter. on the 5' end there is a minor groove binder.


In the Unhybridized state, reporter and quencher will be in close proximity resulting in the quenching of reporter. During the annealing step of pcr probe binds to the target sequence with the help of minor groove binder, once it binds reporter and quencher becomes far apart and fluorescence can be detected. The fluorescent signal is proportional to the amount of amplified product in the sample.

Amplifluor Chemistry

Amplifluor chemistry employ two target-specific primers and one universal primer called the UniPrimer. The first target-specific primer contains a 5' extension sequence called the Z-sequence that is also found at the 3' end of the UniPrimer. The UniPrimer forms a hairpin structure. A fluorescent reporter and a quencher are attached at the 5' and the 3' ends of the stem structure, respectively. In the hairpin conformation, the reporter fluorescence is quenched due to its proximity to the quencher. During the first amplification cycle, the first target-specific primer (with the Z-sequence) hybridizes to the template and is extended. During the second amplification cycle, the second target-specific primer is used to synthesize a new target template that contains a sequence complementary to the Z-sequence. The product from the second amplification cycle can then serve as the template for the UniPrimer. In the third amplification cycle, the extended UniPrimer serves as a template for the next amplification cycle. In the fourth cycle, extension of the template through the hairpin region of the UniPrimer causes the UniPrimer to open up and adopt a linear configuration, which allows the reporter to fluoresce. Exponential amplification using the second target-specific primer and the UniPrimer occurs in subsequent amplification cycles. The resulting fluorescent signal is proportional to the amount of amplified product in the sample. 

Scorpion Primer Assay




Scorpions primer assays employ two primers, one of which serves as a probe and contains a stem-loop structure with a 5' fluorescent reporter and 3' quencher. The loop sequence of the Scorpions probe is complementary to an internal portion of the target sequence on the same strand. During the first amplification cycle, the Scorpions primer is extended and the sequence complementary to the loop sequence is generated on the same strand. The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent read-through during the extension of the opposite strand. After subsequent denaturation and annealing, the loop of the Scorpions probe hybridizes to the target sequence by an intramolecular interaction, and the reporter is separated from the quencher. The resulting fluorescent signal is proportional to the amount of amplified product in the sample.

LUX (Light Upon Xtenstion) Primer Assay

LUX primer assays employ two primers, one of which is a hairpin-shaped primer with a fluorescent reporter attached near the 3' end. In the intact primer, the reporter is quenched by the secondary structure of the hairpin. During amplification, the LUX primer is incorporated into the product and the reporter fluoresces.

LUX Probe Invitrogen Manual

BD QZyme Primer Assay

BD QZyme primers employ a target-specific zymogene primer, a target-specific reverse primer, and a universal oligonucleotide substrate. The oligonucleotide contains a fluorescent reporter on the 5' end and a quencher on the 3' end. When oligonucleotide substrate is intact, the fluorescence of the reporter is quenched by the quencher due to their proximity. The zymogene primer contains a sequence that encodes a catalytic DNA. During the first amplification cycle, the zymogene primer is extended. In the second cycle, the product of the first cycle is used as the template by the target-specific reverse primer, which is extended to create a new target sequence containing a catalytic DNA region. In the subsequent annealing step, the fluorescently labeled oligonucleotide substrate hybridizes to the catalytic DNA sequence and is cleaved. This cleavage separates the reporter from the quencher, resulting in a fluorescent signal that is proportional to the amount of amplified product in the sample. 
References

Minor Groove Binder-Conjugated DNA Probes for Quantitative DNA Detection by Hybridization-Triggered Fluorescence - I.A. Afonina, M.W. Reed, E. Lusby, I.G. Shishkina, and Y.S. Belousov
Epoch Biosciences, Bothell, WA, and Synthetic Genetics, San Diego, CA, USA

Use of self-quenched, fluorogenic LUX primers for gene expression profiling. - Kusser W.

Multiplex real-time PCR assay using Scorpion probes and DNA capture for genotype-specific detection of Giardia lamblia on fecal samples. - Ng CT, Gilchrist CA, Lane A, Roy S, Haque R, Houpt ER.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.- Buh Gasparic M, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, Zel J, Gruden K.

Technical Resources:
Sigma Aldrich
Life Technologies
Biorad Laboratories 
Gene Quantification

Wednesday, December 4, 2013

Protein Quantification / Protein Estimation using Polyacrylamide gel Electrophoresis (PAGE)

The Sodium Dodecyl Sulfate -Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a technique for the characterization of proteins both quantitatively and qualitatively. It is a separation technique to separate out proteins from a mixture based on the molecular weight. SDS- PAGE can also be used quantify  particular proteins at microgram level from a mixture. The quantification analysis can be done by scanning the stained electrophoresed gel by densitometry. If the polypeptides are radiolabelled then it can be visualized and quantified using flourographic plate.

Principle
The percent absorption of incident light is directly proportional to the color intensity of the protein-dye complex on the gel and is directly related to the protein concentration.
Similarly, the intensity of darkening of the X-ray plate is directly proportional to the radioactivity in the protein in fluorographic plates.

Materials
  • A spectrophotometer with suitable scanning facility and chart recorder (or integerator facility, if available)
  • Protein Stain (Quantitative): 0.2% Proceion Navy MXRB dye in Methanol : Acetic Acid : Water (5:1:4). Dissolve the dye first in the methanol and then proceed. Prepare fresh everytime.
  • Destaining Solution: Methanol : Acetic Acid : Water (1:1:8).
  • Fluorograph Plate - For Fluorograph Scan
Procedure
  1. 1. After the electrophoresis (SDS-PAGE of protein), immerse the gel in Proceion Navy dye solution and shake until the proteins are completely stained (for a fixed period ~ 2hrs).
  2. Destain the gel until the background is colorless.
  3. Scan the gel at 580nm to measure the degree of dye bound by each band of protein. Depending upon the type of equipment available for scanning, the whole gel is used or each lane is cut out and scanned individually. The total absorption by the dye in each band is proportional to the area of the peak in the scan profile.
  4. Each peak in the scan profile is traced using a planimeter to determine the area under it. Otherwise, each peak in the chart may be cut out and weighed. When an integrator is interposed, the area under each peak is automatically calculated. 
  5. A curve is obtained by plotting A580 vs. amount of protein used as standard. Bovine serum albumin (Fraction V) at different known concentrations co-electrophoresed in different lanes in the same gel is also used to construct the standard curve. It should however be noted that the protein both in the standard and under examination to have equal dye-binding property.
  6. Scanning Fluorographic Plate
  7. Scan the individual lane strip or the whole fluorographic plate at 620nm as described above. The standard curve is obtaining using a radioactivity labeled standard protein whose concentration and radioactivity are known.
Notes

The following conditions need to be satisfied to quantity proteins on the gels:
  • The protein bands should be well resolved,
  • The dye should be bind to the protein of interest, and the binding should be uniform to all proteins and the sampling errors should be small.
  1. If the peaks are not well resolved, use of the narrower beam of light will improve the situation but at the cost of baseline.
  2. Coomassie brilliant blue R250 staining is not suitable for quantitative analysis of proteins although it is a highly sensitive stain. 
  3. Proceion Navy dye binds to the proteins stochiometrically and covalently. Destaining of this dye from the gel requires longer time.
  4. Sampling errors are inevitable but their effect can be reduced by repetition and averaging the results.
  5. Electrophoresis with a fixed sample volume, voltage and duration of run is necessary between runs to obtain satisfactory results.
  6. Gel scanner with computer facility are now commercially available.
References
1. Carlier, A R, Manickam, A and Peumans, W J (1980) Planta 149 227.
2. Smith, B J, Toogood, C and Johns, E W (1980) J Chromatogr 200 200. 

Wednesday, November 20, 2013

World's Top 10 Bioenergy Companies

Bio-Energy

Bioenergy is the Renewable Energy that is produced from the materials that are derived from biological sources.

What does the Bio-Energy Companies do??

Bioenergy companies focuses on production of renewable energy by safer, cleaner and cheaper ways for the betterment of humans.

Here is the list of Worlds Top 10 Bioenergy Companies




1. Solazyme




Research Areas / Products
  • Renewable Fuels
  • Petrochemicals
  • Nutritionals
  • Health Sciences 


2. KiOR




Research Areas / Products
  • Real Fuels
  • Green House Gas reductions
3. LanzaTech



Research Areas / Products
  • Gas Fermentation
  • Synthetic Biology
  • Microbiology: LanzaTech has one of the world’s largest collections of industrial fuel and chemical production microbes. To date, we have two new proprietary strains of gas fermentation microbe.
  • Analytical Chemistry
  • Fermentation


4. Novozymes




Research Areas / Products
  • Agriculture
  • Bioenergy
  • Biopharma 
  • Food & Beverages
  • Household Care
  • Leather 
  • Pulp & Paper
  • Textile
  • Wastewater Solutions

5. POET


Research Areas / Products
  • BIOFUELS (ETHANOL)
  • FOOD & FEED
  • OIL & LUBRICANTS
  • SPECIALTY CHEMICALS


6. DuPont Industrial Biosciences



Research Areas / Products
  • Animal Nutrition
  • Biomaterials
  • Biofuels
  • Carbohydrate Processing
  • Detergents
  • Food Enzymes
  • Personal Care
  • Safety and Restoration
  • Textiles



7. Gevo



Research Areas / Products
  • Biofuel - Isobutanol
  • Solvents and Coatings
  • Materials, Plastics, and Fibers
  • Biojet Blendstock
  • Specialty Fuels 
8. Sapphire Energy



Research Areas / Products
  • Green Crude

9. Joule Unlimited


Research Areas / Products
  • Biofuel





10. ZeaChem



Research Areas / Products

Cellulosic Ethanol
Acetogens



Reference
Internet Sources

*All Images and videos & Trademarks belongs to the respective owners

Saturday, November 9, 2013

Nanobodies - A new Therapeutic Approach

Nanobodies are type of antibodies derived from camels, smaller than the conventional antibodies. Nanobodies are also known as single domain antibodies.



Antibody Vs Nanobody




Problems Associated with use of Antibodies

Storage – Require Freezing temp.
Oral Administration – digested in gut
Cannot cross Blood-Brain Barrier
 MAb therapies need to be given by injection or infusion at a clinic.

Advantages of Nanobodies
Nanobodies are simpler,
Nanobodies are smaller which perform better,
Nanobodies are easier to make,
Nanobodies are easier to handle,
Nanobodies are easier to admister, and
Nanobodies are more affordable.

Applications of Nanobody

Therapeutical Uses:

Can be used for oral administration.
Oral nanobody pill for inflamatory gut disease and colon cancer.

Papers on Nanobody as Therapeutic Target

Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

Nanobodies as novel agents for disease diagnosis and therapy

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.

Reference

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.

References:

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

Wednesday, September 25, 2013

Immunoprecipitation: Procedure, Analysis and Applications

Immunoprecipitation

Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.

Immunoprecipitaion in general involves the following Steps:
  • Incubate sample with antibody against protein of interest.
  • Separate antibody-protein complex from remaining sample
  • Analysis


Basic Principle Of Immunoprecipitaion

Immuno precipitation


Co-Immunoprecipitation:

A protein complex can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex.

Applications of Immunoprecipitation Technique

  1. Isolate / Detect Proteins of interest
  2. Enrichment of low abundant proteins
  3. Study protein-protein interaction and protein complexes
  4. Identify unknown proteins in a protein complex
  5. Verify protein expression in a specific tissue.
Immunoprecipitaion Procedure / Protocol

Immunoprecipation Procedure / protocols involves the following steps
  1. Sample Preparation
  2. Use of an Isolate control
  3. Pre-Cleaning of sample
  4. Antibody Incubation
  5. Precipitation of Protein / Protein Complex
  6. Washing
  7. Elution
  8. Analysis of the Precipitate
Lets look into each Step:

Sample Preparation


Samples used for immunoprecipitaion can be of any samples of biological origin.
Lysis Buffer: Choice of buffer depends on goal of the immunoprecipitation experiment

Lysis Buffer Criteria
Concentration Range
Type and amount of detergent
Non-Ionic : 0.1 – 2.0 %
Ionic: 0.01% to 0.5%
Amount of Salt
0-1 M
Presence of EDTA
0-5MM
pH
6.0 – 9.0

Lysis Buffer should always contain protease inhibitors and phosphatise inhibitors
Store lysates at -20dc or  -80dc
Avoid freeze thaw cycles

Isotype Control
Isotype control is used to establish the specificity of the signal and the amount of non-specific background. It should be done simultaneously with the IP antibody but in a different tube.

Pre-Clearing
This step is done to avoid any non-specific binding and thereby avoiding the background signal. This step helps to reduce the background and improve signal to noise ratio. It is an optional step to improve the signal.
Antibody Incubation
Amount of antibody required need to be find out and it is important to have optimal antibody to protein ratio to have better results.
Different antibody to protein concentration ratios can be tried out (1:100 to 1:1000), Antibody incubation is done by incubating the IP antibody with the lysate by gentle agitation, it can be done at room temperature for 2hrs or at 4dc overnight . Incubation time and antibody concentration need to be optimized for better results.
Precipitation of Protein / Protein Complex
Protein A,G or L coupled to beads (Aarose or Sepharose) are most commonly used for Protein precipitation. Base on the host species and the type IP antibody beads can be selected.
Antibody can be directly conjugated to the beased the advantage of this is that of having lesser non-specific bands.
Immunoprecipitation is done by incubating with the antibody and then centrifuging it at 4dc.

Washing
Washing is done to remove the non-specifically bound proteins from the immunoprecipitate. Washing is generally  done with Lysis buffer or PBS. PBS is less stringent and can be used for analysis of protein-protein complexes.

Elution
Elution step is to dissociate the specifically bound proteins from antibody-bead complex.
Elution Buffers:
2X Laemmli Buffer: Harsh Buffer can denature protein
Glycine Gradient (upto 1M):  More gentle can dissociate protein of interest without eluting IP antibody.

Analysis of the Precipitate:

Analysis can be done using the following methods
  1. SDS – PAGE
  2. Western Blotting
  3. Gel band Excision and sequencing
  4. Mass Spectrometry
  5. Scintillation counter or X-ray film for radioactive samples, etc.



References:

Abcam Technical Resources
Thermo-Scientific Technical Resources 

Friday, September 20, 2013

Sequence Alignment and Primer Designing Using Bioedit

BioEdit is a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7. An intuitive multiple document interface with convenient features makes alignment and manipulation of sequences relatively easy on your desktop computer. Several sequence manipulation and analysis options and links to external analysis programs facilitate a working environment which allows you to view and manipulate sequences with simple point-and-click operations.




BioEdit's features include:
  • Several modes of hand alignment
  • Automated ClustalW alignment
  • Automated Blast searches (local and WWW)
  • Plasmid drawing and annotation
  • Accessory application configuration
  • Restriction mapping 
  • RNA comparative analysis tools
  • Graphical matrix data viewing tools
  • Shaded alignment figures
  • Translation-based nucleic acid alignment
  • ABI trace viewing, editing and printing

Let's see how to do a sequence alignment using bioedit software....

First thing you have to do is to get the sequences of your interest, it can be whole genome sequence or particular gene.This can be retrieved from NCBI (National Center for Biotechnology Information) .

For Example let's search for Haemagluttin gene (HA) of H1N1

You can save the selected gene sequence in FASTA format, which can be used in Bioedit software.


You can import multiple sequence files into one window
File > New Alignment. File> Import> Sequence alignment file> choose text file that save the FASTA sequences.

Once all the sequence have been selected you can run Clustal W from the Accessory application menu

choose all sequence> Accessory Application> ClastalW Multiple alignment> Run ClastalW> OK> Alignment> Find Conserved Regions> Start




The result will show how many conserved regions found and details of each region. Then choose one region as the template

 


Then use the chosen one region as template for real-time PCR primer and probe design. Before the region is used as template, checking specificity of this region by alignment of this region is required by using “Nucleotide BLAST”

 

Conserved regions are important because these regions will have least mutations, so these are ideal region for designing primers and probe for qPCR.

Primers can be Picked up from the conserved region using one of the many softtwares, to mention few

  • Primer 3
  • Primer Premier
  • NCBI
  • Oligo Designer, etc
References and Links:

Download BioEdit
BioEdit Help Files

Tuesday, September 3, 2013

Top 10 Blockbuster Drug Molecules!!!!

These molecules made the companies billion dollar worth...


Rank: 1



Trade Name: Lipitor 


INN: Atrovastatin

Company: Pfizer 

Technology: Chiral Chemistry 

Therapeutic Use: Cholesterol Lowering 

Sales : $125 Billion

Patent Expired : Novemeber 30, 2011




Rank 2

Trade Name: Plavix 

INN: Clopidogrel

Company: Sanofi/ Bristol 

Technology: Small Molecule Chemistry

Theraputic use: Anticlotting

Sales: $19.5 Billion, Patent Expiry: 2012




Rank 3

Trade Name: Advair

Company: Glaxo Smith Kine

Technology: Small Molecule Chemistry

Therapeutic Use: Asthma

Sales: $9.0 Billion

Patent Expiry: 2013 (European)








Rank 4


Trade Name:  Remicade

INN: Infliximab

Company: Merck,J&J

Technology: Monoclonal Antibody

Therapeutic Use: Arthritis

 Sales: $7.67 Billion, Patent Expiry: 2015




Rank: 5

Trade Name: Enbrel

Company: Pfizer (Wyeth)

Technology: Recombinant Gene Product

Therapeutic Use: Arthritis 

Sales: $7.1 Billion

Patent Expiry: 2012











Rank: 6

Trade Name: Humira

Company: Abbott

Technology: Monoclonal Antibody

Therapeutic Use: Arthritis

 Sales: $6.8 Billion

Patent Expiry: 2016







Rank 7

Trade Name: Avastin

Company: Roche

Technology: Monoclonal Antibody

Therapeutic Use: Cancer

Sales: $6.7 Billion





Rank 8

Trade Name: Rituxan

Company: Roche

Technology: Monoclonal Antibody

Therapeutic Use: Cancer

Sales: $6.1 Billion 











Rank 9

Trade Name: Diovan

Company: Novartis

Technology: Small Molecule Chemistry

Therapeutic Use: Hypertension

Sales: $ 6.0 Billion











Rank:10

Trade Name: Crestor

Company: Astrazeneca

Technology: Small Molecule Chemistry

Therapeutic Use: Cholesterol Lowering

Sales: $5.8 Billion







Tuesday, August 27, 2013

Artificial Ear - An Ear Lab Grown...!!!!!!!!!

Scientists in US has developed human-like ear from animal tissue, it is one of the milestones in the tissue engineering.

The reports says that the lab grown ear has flexibility same as that of the real human ear.

Scientists believe that this technique will one day be useful for the people who are having deformed ears.


As published in the Journal of Royal Society Interface, scientists took tissues from cows and sheep and flexible wire frame which has 3D shape of real human ear. The cells were grown on a titanium wire scaffold that is modeled on the dimensions of a real human ear, taken from CT scans.

Scientists are expecting the process to be ready for clinical trials in next five years.

There are several advances in the field of tissue engineering, developing/ growing organs in lab for replacing the damaged ones.

It is still very early days for this field, but this study represents a step towards being able to engineer replacement outer ear tissue.

Source

BBC News

Tuesday, July 30, 2013

World's Largest Chromatographic Plant for API Production

Novasep's New plant on its Mourenx, France site to produce a large volume commercial API.

Novasep, a leading provider of purification based manufacturing solutions for life science molecules, announces today an investment of EUR 30 million to build in Europe what will be the world's largest chromatography plant used for the production of a large volume commercial API(active pharmaceutical ingredient).

The plant will be built on Novasep's existing Mourenx site in France and will be operational and validated within another few months. Both the development of an advanced purification process and the plant expansion within the challenging timescale are results of the sharp increase in the projected demand for a large volume, highly purified API.

chromatography plant


Novasep has pioneered continuous chromatography for pharmaceutical manufacturing for more than 20 years. Its SMB and Varicol® continuous chromatography technologies are currently used for commercial scale production of several chiral APIs. The new plant, designed by a Novasep in-house engineering team, will include Varicol® systems with 1,200 mm diameter columns operated at up to 70 bars, the largest ever built in the pharmaceutical industry. The plant will enable the production of a highly purified API from a complex mixture. The systems will also integrate sophisticated solvent recovery systems for the recycling of 99.9% of solvents, resulting in both a very cost-effective and environmentally friendly process.

"Demand for Novasep's advanced, purification-based manufacturing capabilities in the life science industries continues to increase as drugs in development and reaching the market become more complex and specific. This necessitates our third, and largest global plant expansion in 2012," said Patrick Glaser, President and CEO of Novasep. "This successful project, the world's largest chromatography plant in the pharmaceutical industry, demonstrates the validity of the Novasep strategy which is based on the combination of synthesis, biosynthesis and purification. We thank our institutional partners for their active support and our shareholders for their strong involvement to enable this transforming project."

Beyond Novasep's commitment, the EUR 30 million investment has been made possible through financial and/or logistic support by the following partners: the association of local authorities of Lacq, the General Council of Pyrénées Atlantiques, the Regional Council of Aquitaine, the Economic Development Bureau, DIRECCTE Aquitaine, the Prefecture of Pyrénées Atlantiques, Total Group, and OSEO, France's innovation partner. The plant will leverage the SOBEGI platform's utility supply.

Sources

Novasep Website
Bioprocess News Channels

Friday, July 26, 2013

Do We Have Enough Sustainable Energy Sources??

Reports claims, within three decades global energy demand will increase by 56%. 

Due to over-consumption, soon there will be oil shortage. how do we cope with the ever growing global energy demands.

Sticking to the sustainable energy strategy and making active contributions to the sustainable energy developments can make big difference in the global energy demands. 

Sustainable energy is the sustainable provision of energy that meets the needs of the present without compromising the ability of future generations to meet their needs. Technologies that promote sustainable energy include renewable energy sources,such as hydroelectricity, solar energy,wind energy, wave power, geothermal energy and also technologies designed to improve energy efficiency.

HYDROPOWER


hydropower


  1. Hydropower or water power is power derived from the energy of falling water and running water, which may be harnessed for useful purposes mainly for electricity.
  2. Hydropower has been used for irrigation and the operation of various mechanical devices,such as watermills,sawmills,textile mills,domestic lifts,power houses and paint making.
  3. Hydro power is a renewable energy source.
  4. Hydropower types: 
Conventional hydroelectric, referring to hydroelectric dams.

Run-of-the-river hydroelectricity,which captures the kinetic energy in rivers or streams, without the use of dams.

Small hydro projects are 10 megawatts or less and often have no artificial reservoirs.

Micro hydro projects provide a few kilowatts to a few hundred kilowatts to isolated homes,villages, or small industries.

Pumped-storage hydroelectricity stores water pumped during periods of low demand to be released for generation when demand is high.

WIND POWER

Wind power is the conversion of wind energy into a useful form of energy,such as using wind turbines to make electrical power,windmills for mechanical power,wind pumps for water pumping or drainage,or sails to propel ships.
  1. Wind power is a renewable energy source.
  2. Types of wind power: 
Wind turbine– a turbine that converts wind energy into mechanical energy.

Windmill– a machine which converts the energy of wind into rotational energy by means of vanes called sails or blades.

wind mill


Windpump– a windmill used for pumping water, either as a source of fresh water from wells, or for draining low-lying areas of land.

Sail– any type of surface intended to move a vessel, vehicle or rotor by being placed in a wind – in essence a propulsion wing.

MARINE ENERGY

marine energy

  1. Marine Energy or Ocean energy refers to the energy carried by ocean waves,tides,salinity, and ocean temperature differences.
  2. The movement of water in the world’s oceans creates a vast store of kinetic energy,or energy in motion.This energy can be harnessed to generate electricity to power homes, transport and industries.
  3. Marinepower can be renewable or non-renewable energy source.
  4. 4.Forms of ocean energy:
RENEWABLE:

Marine current power- The energy obtained from ocean currents.

Osmotic power-The energy from salinity gradients.

Ocean thermal energy The power from temperature differences at varying depths.

Tidal power -The energy from moving masses of water — a popular form of hydroelectric power generation.Tidal power generation comprises three main forms, namely:tidal stream power,tidal barrage power, and dynamic tidal power.

Wave power-The power from surface waves.

NON-RENEWABLE:

Petroleum and natural gas beneath the ocean floor are also sometimes considered a form of ocean energy.

SOLAR POWER


solar power


  1. Solar power is the conversion of sunlight into electricity, either directly using photovoltaics(PV),or indirectly using concentrated solar power(CSP).Its Renewable source of energy.
  2. Concentrated solar power systems use lenses or mirrors and tracking systems to focus a large area of sunlight into a small beam.Photovoltaics convert light into electric current using the photoelectric effect.

Types of Solar energy: 

Solar water heater– Heating water with solar energy.


Solar Electricity-Using the sun's heat to produce electricity.

Photovoltaic Systems– Producing electricity directly from sunlight.

Passive Solar Heating and Daylighting-Using solar energy to heat and light buildings.

Solar Process Space Heating and Cooling-Industrial and commercial uses of the sun's heat.


Solar Impulse - First Solar Powered Flight 

The world’s first solar airplane able to fly day and night powered solely by the sun has flown San Francisco (CA) – Phoenix (AZ) – Dallas (TX) – St. Louis (MO) – Cincinnati (OH) – Washington D.C. (District of Columbia) – New York City over a period of two months.


solar impulse




BIOENERGY

Bioenergy is renewable energy made available from materials derived from biological sources.

biofuel

Types of Bioenergy

Biopower is electricity generated from combustion of biomass,either alone or in combination with coal, natural gas or other fuel (termed co-firing).

Biofuel is liquid,gas and solid fuels produced from two types of biomass materials – plant sugars and starches (e.g., grains), and lignocellulosic materials (e.g., leaves, stems and stalks).Liquid and gas biofuels are produced through fermentation,gasification,pyrolysis,torrefaction,and transesterification conversion technologies.

*This is a guest post from one of the blog reader.

References:
Wikipedia
Internet Sources
Image Source - Google Images

Tuesday, July 23, 2013

Single Use Bioreactor - Types, Advantages & Disadvantanges

A single-use bioreactor (S.U.B) or disposable bioreactor is a bioreactor with a disposable bag instead of a culture vessel made from stainless steel or glass, a single-use bioreactor is equipped with a disposable bag. The disposable bag is usually made of a three-layer plastic foil.

Many mammalian cell based biotech producers are moving to single use bioreactor modules leveraging the advantages of single use bioreactors.

Types of single-Use Bioreactor:
  • Single-use bioreactors use stirrers like conventional bioreactors, but with stirrers that are integrated into the plastic bag.The closed bag and the stirrer are pre-sterilized. In use the bag is mounted in the bioreactor and the stirrer is connected to a driver mechanically or magnetically. 
Single Use Bioreactor


  • Single-use bioreactors are agitated by a rocking motion. This type of bioreactor does not need any mechanical agitators inside the single-use bag.
single use bioreactor
Single Use Bioreactor - Agitated Type



Advantages:
  1. Eliminates validation issues because the cleaning process is avoided. 
  2. Shortens downtime and turnaround time because no cleaning is required. 
  3. Lowers risk of cross-contamination with use of new bags for each run. 
  4. Offers multiple advantages based on the elimination of the cleaning process. 
  5. Decreased operating costs and capital investment: savings on start-up capital costs as well as utilities, space, and labor requirements. 
  6. Cost savings from reduced cleaning needs: less space and stainless steel equipment needed; decreased cleaning validation and decreased use of WFI and cleaning solutions; cleaning and sterilization of bioreactors or process tanks can be ‘outsourced’ to disposable bag suppliers. 
  7. Elimination of the design elements of traditional stainless steel vessels that are dictated by CIP and SIP requirements: validation is reduced because systems are less complex. 
  8. Ease of installation. 
  9. Ease of moving when empty. 
Disadvantages:
  1. Limitation in liquid transfer. 
  2. Scalability is an issue: larger-scale bioreactor bags are needed than are available. 
  3. Expensive to use:repetitive purchases required. 
  4. Performance is not completely proven: new technology. 
  5. Slight increase in variable costs per run 
  6. Disposable bag systems difficult to justify for dedicated products or greater than 10,000L bioreactor scale processes. 
  7. Most of the larger scale facilities already have the investment in the ground for the tanks and the required cleaning validations to support multiple product use. 
  8. Leachables and inability to store hot liquids. 
  9. Potential for puncture. 
  10. Difficulty in moving when full. 
  11. Pressure and temperature sensitivity. 
  12. Disposal costs. 
Progress for Single Use Bioreactor
  • Wide acceptance of bioprocess bags 
  • Single use bioreactors are scalable and performance is comparable to Stainless Steel bioreactors. 
  • 1000L Bioreactor scales can open up opportunitiesin commercial applications. 

List of Commercially available Single Use Bioreactor Systems
  • XDR - Xcellerex 
  • Mobius - Millipore 
  • CelliGen BLU - eppendorf 
  • Hyclone S.U.B - Thermo Scientific 
  • Single Use Bioreactor - Applikon 

Helicase Dependent Amplification...DNA Amplification Without Thermocycler

Helicase Dependent Amplification (HDA) is an invitro nucleic acid amplificatio method similar to Polymerase Chain Reaction (PCR).



HDA works at constant temperature, where as PCR requires a thermal cycler for the amplification.

Nucleic acids can be amplified by various technologies and the product can then be used in research or in diagnostics. The Polymerase Chain Reaction (PCR) dominates the nucleic acid amplification market. However, the requirement for thermocycling in PCR limits its application in certain areas, such as field-testing and point-of-care diagnostics.

Biohelix Corporation has developed Kits based on Helicase Dependent Amplification Technology.

Helicase Dependent Amplification is  an isothermal DNA amplification system that uses a helicase enzyme to unwind double stranded DNA (dsDNA), referred to as Helicase-Dependent Amplification. In HDA reactions, duplex DNA is separated into single strands by a helicase. Compared with the other techniques, this amplification method is closer to nature's method of performing DNA replication and it has the following advantages:


1. Low cost for instrumentation because use of helicase to separate DNA eliminates need for thermocycler.

2. Easy-to-use for assay development (using two primers).

3. Versatile platform: can amplify both DNA and RNA (with a reverse transcriptase) and is compatible with multiple detection technologies.

4. High sensitivity and specificity.

5. HDA is a rapid method of amplifying nucleic acid at isothermal temperature that doesn't require thermal cycler.

Disadvantages

Mass diagniosis from large no. of samples cannot be achieved.

Cost of HDA reagents are costly compared to PCR reagents.

Progress and Developments of HDA Technology

A team of researchers have successfully implemented the HDA technology for amplyfing the target DNA in combination with an integrated disposable device for DNA extraction. Full Publication can be found in the below link.

An integrated disposable device for DNA extraction and helicase dependent amplification.

http://www.ncbi.nlm.nih.gov/pubmed/20066496

Monday, July 22, 2013

Tangential Flow Filtration: Membrane Types and Process Parameters

Tangential Flow Filtration [TFF] is used for clarifying, concentrating and purifying proteins.

Filtration is a pressure driven separation process that uses a membrane to separate components in liquid solution or suspension based on size and charge difference.

Types of Membrane Filtration




1. Normal Flow Filtration - Dead End Filtration
2. Tangential Flow Filtration - Cross Flow Filtration

Tangential Flow Filtration:

Tangential Flow Filtration is divided into categories based on the size of the components being separated.

Generally membrane pore size is given as micron value, particles sized above this value will be retained.

Types of TFF

1. Micro Filtration 
2. Ultra Filtration

Ultra Filtration is catagorized into two 
  • Virus Filtration
  • High Performance Tangential Flow Filtration
Process Goals
  1. Final Product Concentration
  2. Feed Volume Reduction
  3. Extent of Buffer Exchange
  4. Contaminant Removal Specification
Primary Protein Processing Goals
  1. High Product Yield
  2. High Product Quality
  3. High Product Purity
  4. Controlled Bioburden
Membranes
  • Polyethersulfone (PES)
  • Regenerated Cellulose
Types of Membranes
  1. Flat Plate
  2. Spiral Wound
  3. Hollow Fiber
Key Parameters to Optimize
  1. Cross Flow Rate
  2. Transmembrane Pressure
  3. Filtrate Control
  4. Membrane Area
  5. Diafiltration Design
Factors affecting Product Yield

These are the four contribution of product loss during Tangential FLow Filtration
  1. Retention Loss
  2. Adsorption Loss
  3. Solubility Loss
  4. Unrecoverable hold up volume loss
Factors affecting Product Quality

Quality of product is compromised due to aggregation or denaturation caused by
  1. Micro cavitation
  2. Air-Liquid Interface
  3. High Protein Concentration
  4. Temperature Effects