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Sunday, December 14, 2014

Enzyme Assay - Enzyme Denaturation Mechanism and Enzyme Stabilization Strategies

Enzyme Assay

The role of enzyme assay is to
  • To Identify an Enzyme - Presence or Absence - By qualitative approach.
  • To check amount of enzyme activity - By quantitative approach.

Enzyme identification by catalytic action of enzyme. Enzyme activity depends on the following factors:

pH, Nature and Strength of ions.

General Considerations for an enzyme assay

  • Instability and Instruments
  • Measurements in turbid solutions
  • Contamination
Turbidity caused by weakly soluble substance, soiling, dust or air bubble.

Low scattering only possible, only if the observed component produces a signal (eg: absorption), while other component show no signal (No absorption) in the observed range.

Methods for Observing enzyme reaction

By measuring the appearance or disappearance of colored compound:

  1. Photometry
  2. Fluorometry
  3. Turbidometry
  4. Luminometry
Continuous Assay

Continuous assay is important for determination of enzyme velocity and for evaluating the enzyme activity.

Continuous assay helps in the detection of erroneous influences and artifactual disturbances and control of reaction course (Progress Curve).

Influence of pH on Enzyme assays

Enzyme activity follow bell curve

Two aspects are responsible for pH dependent activity:
  • State of protonation of functional group of amino acids and co-factors involved in the catalytic reaction.
  • The native, 3-D Protein structure of enzyme.
Protonation is reversible, but damage to the protein structure is irreversible.

The Inflexion points of the curve at half maximum velocity (Vmax/2) indicate the pKa value approximately. pH <3 or="">11 even for short time should be avoided except for the enzymes like trypsin which is tolerant to acidic pH.

Influence of Buffer Ions on Enzyme activity

Buffers helps to adjust and stabilize pH during enzyme assay.

pH = pKa - log [HAc] / [Ac-]

pH = -log[H+]; pKa = -log Ka

Two criteria for buffers:
  • Ionic Strength and Concentration : generally 0.05 - 0.2 M used.
  • Nature of buffer components
Influence of Temperature on Enzyme activity

Typical dependence of the enzyme activity on the temperature.
 (A)Direct plotting and(B)Arrhenius diagram.
The green lines represent the range of the increase of the reaction velocity with the temperature;its continuation(dotted violet line) is interrupted by progressive inactivation (redlines).

Inactivation is forced by pre-incubation of the enzyme at the high temperature,causing a decrease and shift of the temperature maximum to the lower range (black arrows).In(A)the three most commonly used assay temperatures are indicated.

Enzyme Denaturation Mechanism

Enzyme denaturation is the unfolding of enzyme tertiary structure to a disordered polypeptide in which key residues are no longer aligned closely enough for continued participation in functional or structure stabilizing interactions.

Invitro Protein Stability:
  1. Thermodynamic Stability (Kinetic Stability)
  2. Long term stability (Conformational Stability)
Enzyme Inactivation Studies:
  • Biochemical / Structural - Effect of temperature and other reagents on secondary and tertiary structure.
  • Mathematical Simulations - Effects of agents on enzyme activity.
Enzyme Inactivation:

Enzyme inactivation is a two step Process

1. Reversible unfolding of original enzyme followed by
2. Kinetically irreversible steps: Aggregation or covalent changes in the enzyme.

Factors Affecting Enzyme Stability / Inactivation
  • Temperature
  • Pressure
  • Salt
Enzyme Stabilization: In aqueous environment
  • Screening enzymes from extremophiles & their isolation.
  • Production of stable enzyme in mesophilic organisms.
  •  Stabilize unstable enzyme by protein engineering, chemical modification, Immobilization and medium engineering by additives.
Extremophiles: Eg: Taq Polymerase in thermus aquaticus

Observation in thermophilic enzymes: 

In thermophilic enzymes lysine is replaced by arginine, Thermophilic enzymes generally have high proline content and aspargine/glutamine content is low.

Thermozymes have more interactions:
  • Hydrogen bonds
  • Disulfide bonds
  • Hydrophobic Interaction
  • Electrostatic Interaction
  • Superior conformational structure.
Kinetic Stability is expressed as half life at defined temperature.

dG is 5-20 kcal/mol.

Modification of Mesophilic Enzymes

Introduction of mutation: Entropic Stabilization by introduction of prolines or disulfide bridges.

Thermal stability due to rigid conformation and higher number of hydrophobic interactions.

Aspargine is thermoliable, threonine or isoleucine have similar geometry to aspargine but are moste heat stable.

Chemical Modification:

Chemical modification of the amino acid side chain can yield stability.
  • Monofunctionally substituted proteins:
Thermostability can be achieved by replacing lysine or histidine by arginine resulting in enhanced intramolecular or inter-subunit salt bridges.
  • Grafting to Polysaccharides
  • Grafting to Polymers
Enzyme Immobilization

Thermal Stability from molecular rigidity introduced by attachment to rigid support and protection by microenvironment.
  • Adsorption
  • Covalent Bonding
  • Entrapment
  • Membrane Confinement
Addition of ligands, salts, etc can give enzyme stability.

  • Ion Effect of divalent cations on thermo-stability. Ions employed in concentration ≤ 0.1 M.
  • Non- Specific ion effect:  Salts employed at higher concentration ≥ 0.1 M. 
Non-specific ions bind to charged groups or dipole leading to salting out resulting in the compression of the enzyme.


Enzyme stability and stabilization—Aqueous and non-aqueous environment Padma V. Iyer, Laxmi Ananthanarayan.

Enzyme assays Review, Hans Bisswanger

Saturday, November 22, 2014

DNA Microarray – Types, Strategies and applications

DNA microarray is a hybridization technique performed in microscopic glass slide with surface modification, anchoring specific set of probes sequences complementary to the Target. Upon complementation with the target sequence fluorescence signal can be captured and state of the test sample can be identified.

DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs), or to genotype or targeted resequencing.

Principle of DNA Microarray

The principle behind microarray is the hybridization between two DNA strands. Complementary sequence of the DNA pair specifically by forming hydrogen bonds, the more number of complementary base pairs in a nucleotide sequence will have a tighter non-covalent bonding between the two strands.

DNA hybridization

After the hybridization of target DNA to the probes attached on the glass slide, slides are washed to remove any non-specifically bound sequences, only strongly paired strands will remain hybridized.

Fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.

Preparing Glass Slide for Surface Modification

Glass slide used for surface modification microarray need to be clean, to remove any organic contaminats and dirt glass can be cleaned with Ethanol, Toulene or Etching with pirhana. Etching with pirhana (7:3 H2SO4:H2O2) is the better choice among  the other cleaning methods.  One need to be careful while while handling pirhana solution as it is a strong oxidizer.

Surface Modification Strategies

 Various Surface modification are developed for DNA microarray, few of the most commonly used ones are

  • Poly – L- Lysine,
  • 3-aminopropyltrimethoxysilane (APS),
  • 3-glycidoxypropyltrimethoxysilane (GPS) and 
  • Aldehyde or carboxylic acid.

Oligo attachment to the modified glass surface

  • Oligo attachment to PLL coated glass slide

Poly – L – Lysine hydrogen bonding with an oligonucleotide.

  • Oligo attachment to APS coated glass slide

3-aminopropyltrimethoxysilane (APS) hydrogen bonding with an oligonucleotide.

  • Oligo attachment to GPS coated glass slide

3-glycidoxypropyltrimethoxysilane (GPS) covalently bound to an amine-terminated oligonucleotide.

  • Oligo attachemnt to DAB Dendrimer modified glass slide

One-half of a DAB dendrimer hydrogen bonding with an oligonucleotide

Spotting Oligo Probes on the glass slide

  • Mechanical Spotting
  • Ink Jetting
Microarray Workflow

Microarray Detection / Capture

List of commercially availbale DNA Microarrays
  • ACLARA Bio Sciences 
  • GeneChipTM - Affymetrix 
  • LabChip - Caliper Technologies 
  • LabCD system - Gamera Bioscience 
  • Genetix Ltd 
  • Agilent 
  • Illumina 
  • Incyte Microarray Systems 
  • Nanogen 
  • Sequenom and 
  • GenoSensor - Vysis Inc. (Downers Grove, IL)

Technical Resources, Array It Corp
Technical Resources, Affymetrix
A Beginer's Guide to DNA microarrays

Saturday, November 15, 2014

Comparison of TAE and TBE Buffers used in Gel Electrophoreis - Advantages & Disadvantages

In molecular biology, TBE and TAE buffers are used for agarose and polyacrylamide gel electrophoresis.

TAE – Tris Acetic acid EDTA.
  • Common buffer used in labs for DNA agarose gel electrophoresis. 
  • TAE is used at pH 8.0. 
  • Generally TAE is Prepared as 50X Stock.

TBE – Tris  Boric Acid EDTA. 
  • Common buffer used in labs for DNA agarose gel electrophoresis. 
  • TBE is used at pH 8.3. 
  • Generally TBE is Prepared as 10X Stock. 

Comparison of TAE Buffer Vs TBE Buffer

Resolution : 

TAE is Good for separating Long DNA Fragments where as TBE is good for separating small / short DNA fragments.

Enzyme Compatibility (Downstream applications like Cloning) :

TAE is the preferred choice if the DNA is used for cloning or other downstream applications. TBE should not be used if the DNA is further used in cloning as the borate is strong inhibitor of many of the enzymes.

Buffering Capacity : 

TAE has low buffering capacity compared to TBE. TBE is stable and has high buffering capacity.

Migration of DNA:

In TAE buffer migration of Linear Double stranded DNA migrates faster. In TBE migration of DNA is slower when compared to the TAE. 


When considering cost TAE buffer is inexpensive compared to TBE.

Thursday, November 13, 2014

DNA Topology - Enzymes and Modes of action on cccDNA

Twist Number:

Twist number is equal to the number of times one strand of covalently closed circular DNA (cccDNA) crosses in front of the other strand.

For right handed helices Tw > 0

Writhing Number:

Writhing number is equal to the number of times the double helix crosses over the long axis of the double helix in 3D space.

Types of Writhe

  • Interwound Writhe
  • Spiral Writhe

Interwound Writhe

For right handed interwound turn Wr < 0 and or left handed interwound turns Wr > 0

Spiral Writhe
For right handed interwound turn Wr > 0  and for left handed interwound turns Wr < 0

Linking Number = Twisting Number + Writhing Number

  • Topoisomerases I
  • Topoisomerases II

Topoisomerases I

Type I Topoisomerases increases the linking number one step at a time.

Topoisomerase II 

Type II Topoisomerases increases the linking number two steps at a time.

DNA gyrase

Special type of topoisomerase II found in prokaryotes which decreases linking number by 2.
DNA gyrase requires ATP for activity.

Saturday, September 20, 2014

Reverse Transcriptases used in RT-PCR

Reverse Transcription is a process in which RNA is converted to cDNA (complementary DNA). The enzyme responsible for reverse transcription is called Reverse Transcriptase.

The Retroviral Reverse Transcriptases has 3 major activities:
  1. RNA-dependent DNA Polymerase. 
  2. Ribonuclease H (RNase H) activity. 
  3. DNA-depedent DNA Polymerase. 
Three forms of RNA-dependent DNA polymerases are mostly used in the invitro synthesis of complementary DNA from RNA template.

1. Mesophilic enzyme encoded by Avian Myeloblastosis Virus (AMV)

2. Moloney Strain of Murine Lukemia Virus (Mo-MLV)

3. Thermostable Tth Taq DNA Polymerase.

Avian Myeloblastosis Virus (AMV) Reverse Transcriptase

The enzyme requires an RNA or DNA template with RNA or DNA Primer have 3’ Hydroxyl group. The enzyme lacks 3’-5’ exonuclease activity, because of this is it prone to introduce error.

The enzyme encoded by AVM shown have high RNase H activity, digests RNA moiety of RNA DNA hyrid and can cleave at template near 3’ terminus of the growing DNA strand if the reverse transcriptase pause synthesis.

The high RNase H activity of AMV can suppress the cDNA yield and restricts its length.

Moloney Strain of Murine Lukemia Virus (Mo-MLV) Revrese Transcriptase
Similar to AMV, Moloney Strain of Murine Lukemia Virus (Mo-MLV) also requires an RNA or DNA template with RNA or DNA Primer have 3’ Hydroxyl group. The enzyme lacks 3’-5’ exonuclease activity, because of this is it prone to introduce error.

RNase H activity of Mo-MLV is weak compared to the AMV, so it’s a better choice for use in RT-PCR.

Mo-MLV RTase reaches maximum activity at 37dC, where as AMV RTase reaches maximum activity at 42dC.

Theremostable Tth DNA polymerase
Tth DNA polymerase is encoded by Thermus thermophilus and the enzyme posses reverse transcriptase activity in the presence of Mn2+. Tth DNA polymerase has the advantage that the same enzyme can be used in both reverse transcription and amplification in same reaction tube.

The main disadvantage of Tth DNA polymerase is that the average size of the cDNA synthesized is 1-2Kb. The use of Mn2+ can lower the fidelity of DNA synthesis.

Commercially available Modified Reverse Transcriptases
Wild type enzyme is modified to yield improved performance. This includes improved transcription rates and longer cDNA strand synthesis. The modified enzymes are active even at 50 dC.

List of Commercially avilable Reverse Transcriptases:
  • RevertAid Reverse Transcriptase, Thermo Scientific 
  • SuperScript® Reverse Transcriptase, Life technologies 
  • StrataScript® Reverse Transcritase, Stratagene 
  • M-MLV Reverse Transcriptase, Promega 
  • M-MLV Reverse Transcriptase, Sigma 
Technical Resources : Invitrogen
Technical Resources : Sigma
Technical Resources : Promega
Technical Resources : Thermo Scientific
Molecular Cloning, A Laboratory Manual, Sambrook

All company and/or product names may be trade names, trademark's and/or registered trademark's of the respective owners with which they are associated.

Tuesday, September 2, 2014

DNA Regulatory Sequence Analysis / DNA Binding Protein Analysis

Gene regulation is a tightly controlled and regulated process. Gene regulation is complex process and involves various control checkpoints. Promoters, enhancers, DNA binding proteins plays major role in gene regulation.

         Transcription of gene is controlled / regulated by Promoter and enhancers.

         The activity of enhancers and promoters are controlled by Transcription factors (the DNA binding Proteins).

         The DNA binding proteins plays a major role in the gene regulation, which can be identified by the following methods.

Methods for Identifying DNA binding Proteins / Gene Regulation

Following are some of the methods used for identification and analysis of gene regulation

         DNA Foot printing – Identifies site at which the protein binds.

         Gel Shift Assay – Identifies DNA-Protein Complex.

         CAT Assay – Measures Transcriptional Activity.

DNA Foot Printing

In DNA Foot Printing method,

The 5’ end of cloned DNA fragment having the promoter or enhancer to be studied is radiolabeled. 

The labeled DNA fragments are then divided into 2 sets. The first is set is incubated with nuclear extract having DNA binding proteins and the second set is used as control without incubating. 

After the incubation, both sets are treated with nucleases. Nucleases cleave the DNA into smaller fragments. 

Nucleases cannot cleave the regions where the protein is bound. The nuclease treated products are run on gel and autoradiographed.

By comparing the migration patterns of control Sites at which the DNA binding proteins interacts can be identified. The X-ray film gives the DNA foot print.

Gel Shift Assay

In Gel Shift Assay method,

DNA migration under electric field is used to identify the DNA binding proteins interaction. The migration of DNA-Protein complex is much lesser than the DNA alone. The radiolabeled DNA fragments are incubated with nuclear extract having DNA binding proteins, after incubation DNA fragments are run on gel along with the control (Not Incubated).

 By looking at the autoradiographic X-ray film DNA-Protein complex can be identified.

CAT Assay

In CAT Assay method,

Reporter gene is cloned with the promoter to be studied. The promoter-reporter construct is introduced into the cell for analyzing the transcriptional level. Active Promoter will initiate transcription of the reporter gene and the level of product formation can be analyzed.

Mostly used reporter genes:

         CAT gene – Chloramphenicol Aceyl Transferase.

         Luciferase gene.

         Expression levels of these genes are used to analyze the promoter.


Lehniger, Biochemistry
J.D Watson, Recombinant DNA
Kuby, Immunology

Sunday, August 10, 2014

Serial Dilution of Bacterial culture / Stock solution - Method and Calculation

Serial Dilution is a series of sequential dilution of a substance in solution.

How to do serial Dilution?

To dilute a stock solution, Mix the stock solution with diluent. The ratio of the Final volume to the aliqout volume of the stock is known as Dilution Factor (DF). Dilution is the inverse of Dilution Factor. 10 fold dilution is actually 10^-1 dilution (Inverse of Dilution Factor: 1/10).


Diluting a 1 M (Molar) Stock solution 1000 fold by serial Dilution.

1000 fold serial dilution can be done by three 10 fold dilutions. (10*10*10 = 1000 fold).

Lets see how to serially dilute 1 M stock solution to a final volume of 100 mL.

Dilution Factor = Final Volume / Volume of Stock.

Since we need to do 10 fold dilution, dilution factor is 10.

Final Volume - 100 mL
X - Stock Solution required.

so; 10 = 100 mL /X mL;

10X = 100; X= 100/10 = 10 mL.

Diluent Volume required = [Final Volume - Stock Volume] = 100mL - 10mL = 90mL.

To do a 10 fold dilution of 1M Stock solution to 100mL,

Mix 10mL of 1M stock with 90mL of Diluent. Label the tube as Dilution 1.

From Dilution 1 tube take 10mL and mix 90mL of diluent and Label it as Dilution 2.

From From Dilution 2 tube take 10mL and mix 90mL of diluent and Label it as Dilution 3.

As we have done 10 fold dilutions, the tube 3 (Dilution 3) will be 1000 fold diluted than the stock.

Stock solution is 1 M, after 1000 fold dilution it becomes - 1/1000 = 0.001 M.

Dilution 1 - 0.1 M
Dilution 2 - 0.01 M
Dilution 3 - 0.001 M

How to do Serial Dilution of Bacterial Culture?

Dilution of bacterial culture can be performed the same way as explained above. Serially dilute the bacterial culture, Bacterial culture is generally serially diluted for plating (for bacterial isolation), for re-inoculation or for other applications like checking optical density, etc.

Below is the Overnight incubated plate, plated by serial dilution of the sample. Note the plate with lesser number bacterial colonies that is the one which is plated from a more diluted solution.

To calculate CFU/mL or CFU/g Click here

Bacterial Culture Dilution & Calculation

You have a bacterial culture, which has 10^6 bacterial cells  and you want 100 cells/mL how to dilute the culture to get 100cells/mL

Inital Concetration - 10^6 cells
Final Concentration Required - 100 cells/mL or 10^2 cells/mL

You need to do Four 10 fold dilution [10*10*10*10 = 10000 fold dilution] to a volume of 1mL to get  10^2 cells/mL final concentration.

here is how you can do it,

Stock of Bacterial Culture - 10^6 Bacterial Cells

Tube 1 (Dilution 1) - Take 900uL of sterial water in tube 1 and add 100uL of bacterial stock - so tube 1 will have10^5 cells/mL.

Tube 2 (Dilution 2) - Take 900uL of sterial water in tube 2 and add 100uL from Tube 1 - so tube 2 will have10^4 cells/mL.

Tube 3 (Dilution 3) - Take 900uL of sterial water in tube 3 and add 100uL from Tube 2 - so tube 3 will have10^3 cells/mL.

Tube 4 (Dilution 4) - Take 900uL of sterial water in  tube 4 and add 100uL from Tube 3 - so tube 4 will have10^2 cells/mL or 100 cells/mL.

Serial Dilution Calculation: Problems and Solutions

You do a dilution by combining 100 ml volume of NaCl plus 700 ml unit volumes of Distilled water.  What is the dilution factor, i.e, how many more times dilute is it than the original concentration?


Final Volume = Volume of Diluent (Distilled Water here) + 100 (Volume of Stock ) = 800 mL

Dilution Factor = Final Volume / Volume of Stock Aliquot

                        = 800 / 100 = 8

Dilution Factor is 8 or the stock is 1/8 times diluted.

Thursday, July 31, 2014

Yeast Two Hybrid System - For Protein - Protein Interaction Studies

Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.

The Yeast Two Hybrid system makes use of modular nature of gene activator proteins. These proteins bind to the DNA and activate the transcription of the gene.

How the Yeast Two Hybrid System Works

Even though it looks complex, Yeast Two Hybrid System is relatively simple to use in labs to study protein-protein interaction. As the protein-protein interaction occurs inside the yeast cell nucleus, proteins from every part of the cell and from any organism can be studied using yeast two hybrid system.

This is how the Yeast Two Hybrid System works:

The DNA sequence that encodes target protein is fused with the DNA that encodes the DNA-binding domain of gene activator protein using recombinant DNA technology.

Two sets of proteins are used:

·         Bait and the Prey

Target protein is fused to a DNA-binding domain that localizes it to the regulatory region of a reporter gene as “bait.”

Specially designed protein in the cell nucleus (“prey”).

Bait can bind to the regulatory region of the reporter gene, which acts as a bait and can be used to fish out the protein that interact with the target protein inside yeast cell. The interaction of Bait and Prey will bring about the activation and expression / transcription of reporter gene. The reporter gene is the one which will help the yeast cell grow on selective medium.

Many potential binding partners can be prepared by ligating DNA encoding the activation domain of gene activator protein to a large mixture of DNA fragments from a cDNA library.

Cells that express this reporter are selected and grown, and the gene (or gene
fragment) encoding the prey protein is retrieved and identified through
nucleotide sequencing.
Through Two Hybrid System a protein linkage map has been generated for most of the 6,000 proteins in yeast and similar projects are underway to catalogue the protein interactions in C. elegans and Drosophila.

A similar technique called reverse two hybrid system, which can be used to detect mutation of chemical compounds that are able to disrupt specific protein interactions. In reverse two hybrid system the reporter gene can be replaced with a gene that kills the cells when the bait and prey proteins interact. Eliminating a particular molecular interaction can reveal something about the role of the participating proteins in the cell. In addition, compounds that selectively interrupt protein interactions can be medically useful: a drug that prevents a virus from binding to its receptor protein on human cells could help people to avoid infections.

Alberts, Molecular Biology of the Cell
Cooper, Molecular Biology of the Cell
The Yeast Two-hybrid System - Paul L. Bartel, Stanley Fields

Monday, July 28, 2014

DNA Sequencing - Traditional and Next gen Sequencing Methods

DNA sequencing is the process of finding out the exact order of nucleotide present in the DNA molecule. DNA sequencing has revolutionized medical and biological research. Before going into the details of DNA sequencing let us have some basics on the DNA molecule.

Central Dogma of Molecular Biology

Nucleic Acid - DNA / RNA Overview

Nucleic acids are first discovered in 1869 by Miescher. Found as a precipitate that formed when extracts from nuclei were treated with acid. Compound contained  C, N, O, and high amount of P.since it was an acid compound found in nuclei therefore named nucleic acid.

DNA / RNA are made up of nucleotides, these are the building blocks. Nucleotides composed of a Nitrogenous base, a pentose sugar and a phosphate group.

DNA has Adenine,Thymine,Guanine  and Cytosine, where as in RNA the nitrogenous bases found are Adenine,Guanine, Cytosine and Uracil.

The sugar present in DNA is deoxy ribose, where as in RNA it is D-ribose. Thats the reason DNA is called as 2-deoxy ribo nucleic acid.

A nucleotide comprises of a Pentose Sugar, Nitrogenous Base and a Phosphate Group.

The arrangement of Nucleotide polymer in DNA and RNA is as follows:

In DNA Adenine pairs with Thymine by a double bond and Guanine pairs with Cytosine by triple bonds.

DNA Sequencing: 
  • Maxam Gilbert Method of DNA sequencing
  • Chain Termination Method / Sanger's Sequencing Method
Maxam Gilbert Method of DNA sequencing

Sanger's Sequencing

Sanger's sequencing method was developed by Frederick Sanger and colleagues. This method is also know chain termination method. Sanger's sequencing method requires single stranded DNA template, Primer, Taq DNA polymerase, dNTPs and modified nucleotides namely ddNTPs (dideoxy nucleotide triphosphate). when a ddNTP is incorporated into a growing chain, the chain terminates. ddNTPs are usually flourescently / radiolabelled. By looking at the emission of light from the ddNTP sequence of the DNA can be deduced. the sequencing product will be of varying lengths which can be separated by Gel / capillary electrophoresis to read out the base sequence.

Next Gen Sequencing Methods:
  • Ion Torrent Sequencing


  • Pyrosequencing

  • SOLiD Sequencing

  • Illumina Sequencing Technology



Illumina Technical Resources
Applied Biosystem, Technical Resources
Other Internet Sources

Saturday, June 14, 2014

The Cell Cycle

Cell Cycle 

Cell Cycle is the process by which a cell and its contents divide, to give rise to two daughter cells and this process is known as mitosis.

 The Cell Cycle describes the different phases through which the cell divides. Cell cycle occurs through 4 different phases. These are

cell cycle Phases

  • G1 Phase
  • S Phase
  • G2 Phase
  • M-Phase
 The two major events occurs during cell cycle is the DNA synthesis and the chromosome segregation. DNA synthesis occurs in the S-Phase of the cell cycle; in this step the entire DNA (genetic content) of the cell is replicated. In M-phase or the mitotic phase chromosome segregates.

Between the S-Phase and M-Phase there are two checkpoints or Gap Phases namely G1 and G2 Phases. These are the checkpoints or error check phases where the cell confirms the proper replication of the DNA and the energy requirements are met to proceed to the next phase.

Cell Cycle - S-Phase

The major events that occur in the S-Phase or Synthesis phase of the cell cycle are

  • DNA Replication and
  • Sister chromatid cohesion
 DNA replicates and gives rise to chromatids and a pair of chromatids are called sister chromatids, these are held together by a protein called cohesin and the process is called as sister chromatid cohesion.

Cell Cycle - M - Phase

After the DNA replication process, the cell enters the checkpoint G2 Gap Phase, where necessary error checks are done and proceeds for the M – Phase or the mitosis phase. During the M-Phase the sister chromatids moves to the poles of the cells and the cell divides. Each daughter cell receives the identical genetic material.

Mitosis Phase – Overview

Mitosis Phase of cell cycle occurs through 4 different phases:

  • Prophase
  • Metaphase
  • Anaphase and
  • Telophase      


Prophase - Key Events
  • Chromosome Condensation
  • Nuclear Membrane Breaks
  • Microtubule organizers moves to the poles
Metaphase -  Key Events
  • Attachment of Microtubule to the centromere
  • Chromosomes are arranged to the center of the cell
Anaphase -  Key Events
  • Microtubules pulls the chromatids to the poles
Telophase -  Key Events
  • Nuclear membrane formation around the chromosomes
Cell divides by process called cytokinesis, where the cytoplasm of the two cells physically separates giving rise to 2 daughter cells.

Monday, May 26, 2014

Estimation of free fatty acids

Fatty acids are carboxylic acids with a hydrocarbon chain. They have a chain of even number of carbon atoms, generally 4-28. Fatty acids are of Two Types:

  1. Saturated Fatty acids
  2. Unsaturated Fatty acids
Saturated fatty acids have carbon - carbon single bond, whereas unsaturated fatty acids have carbon-carbon double bond.
Structure of Fatty Acid

Fatty acids are usually derived from triglycerides or phospholipids. when they are not attached to other molecules it is called as Free Fatty acids.

A small quantity of free fatty acids is usually present in oils along with the triglycerides. The free fatty acid content is known as acid number/acid value. It increases during storage. The keeping quality of oil therefore relies upon the free fatty acid content.


The free fatty acid in oil is estimated by titrating it against KOH in the presence of phenolphthalein indicator. The acid number is defined as the mg KOH required to neutralize the free fatty acids present in 1g of sample. However, the free fatty acid content is expressed as oleic acid equivalents.

  • 1% phenolphthalein in 95% ethanol.
  •  0.1N potassium hydroxide
  • Neutral Solvent: Mix 25mL ether, 25mL 95% alcohol and 1mL of 1% phenolphthalein solution and neutralize with N/10 alkali.
  1. Dissolve 1-10g of oil or melted fat in 50mL of the standard solvent in a 250mL conical flask.
  2. Add a few drops of phenolphthalein.
  3. Titrate the contents against 0.1N potassium hydroxide.
  4. Shake constantly until a pink color which persists for fifteen seconds is obtained.

Acid value (mg KOH/g) = (Titrate value x Normality of KOH x 56.1) / Weight of sample (g)

The free fatty acid is calculated as oleic acid using the equation
1mL N/10 KOH = 0.028g oleic acid.

Saturday, May 3, 2014

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide (EtBr) is an intercalating agent widely used in molecular biology labs as a fluorescent dye for nucleic acid gel electrophoresis .Ethidium Bromide is very sensitive in detecting DNA bands on gel.

etbr structure
Structure of Ethidium Bromide (EtBr)

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide contains tricyclic phenanthridine ring system that is able to interact with stacked base pairs of double stranded DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and due to that it can bind to the hydrophobic interior of the DNA molecule. The peripheral phenyl and ethyl groups projects into the major groove of DNA helix.

intercalation of EtBr with DNA

Approximately one ethidium bromide molecule will get intercalated per 2.5 base pairs, there will be a 20 fold increase in the fluorescence when intercalated. Absorption maxima of EtBr is in the UV range, upon excitation EtBr emits orange light with a wavelength of 605 nm.

EtBr Stained DNA gel / Agarose gel
EtBr Stained Gel

Ethidium Bromide can bind to helical regions formed by  intrastrand base pairing in RNA and in heat denatured or single stranded DNA. The fluorescent yield of DNA-Ethidium Bromide dye complex is many fold higher than the free dye, so it is advantageous in visualizing the DNA on gels. small amounts of DNA ~10ng can be detected using EtBr staining. Ethidium Bromide stock solutions are prepared at a concentration of 0.25  - 1.0 microgram /mL for gel staining.

Ethidium Bromide is considered as a potent mutagen,  it is called as intercalating agent since it can intercalate into the DNA and can interfere with DNA replication and Transcription.

Safer Alternatives
There is still controversies regarding the toxicity of Ethidium Bromide. Even then some researchers prefers to use Ethidium Bromide for nucleic acid staining. But there are some safer alternatives to Ethidium Bromide, here are the commercially available safer dyes: 
  • SYBR Safe, 
  • Gel Red

Sunday, April 27, 2014

Hydrophobic Interaction Chromatography - Theory and Principle

Hydrophobic Interaction chromatography, is a powerful technique for the separation and purification of biomolecules. Hydrophobic Interaction Chromatography is widely used for the separation and purification of proteins in their native state, as well as for isolating, protein complexes and studying protein folding and unfolding.

HIC was initially termed as salting out. Hydrophobic Interaction chromatography,utilizes the reversible interaction of protein and the hydrophobic ligand for the separation of protein mixtures.

Hydrophobic Interaction Chromatography (HIC) Mechanism

Proteins separates out based on the increasing order of hydrophobicity.

Proteins containing hydrophobic and hydrophilic regions are applied to an HIC column, in a high-salt buffer.

The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that, become exposed are adsorbed by the media.

The more hydrophobic the molecule, the less salt is needed to promote binding.

Usually a decreasing salt gradient, is used to elute samples from the column, in the order of increasing hydrophobicity.

Sample elution, may also be assisted by the addition of, mild organic modifiers or detergents to the elution buffer.

Hydrophobic Interaction Chromatography Theory

There are three major theories, explaining the mechanism of hydrophobic interaction chromatography. They are
  • Salting Out and Hydrophobic Interaction Theory,
  • Thermodynamic Theory, and
  • Surface Tension, van der Waals Forces Theory.

Salting Out and Hydrophobic Interaction Theory

The elution or precipitation strength, of an ion is described by the Hofmeister series. Small, highly charged ions are strong precipitators, whereas organic acids and bases, have a more stabilizing effect, in the presence of proteins in solution. The term chaotropic, refers to the ability of the ion to produce chaos in the water structure.

salting in and salting out

Anti chaotropic salts, such as ammonium sulphate and sodium sulphate, expose hydrophobic patches on proteins by removing the highly structured water layer, which usually covers these patches in solution. As a result hydrophobic residues on a protein molecule can interact with the hydrophobic ligands of the matrix. Salts can also reduce the solubility of proteins by shielding charged groups which normally keep proteins apart in solution. When the electrostatic charge on protein molecules are shielded, the molecules can easily interact, form aggregates and eventually precipitate. The solubility, of different proteins is reduced to different extents by salt addition.

hofmeister series salts HIC

Chaotropic agents disrupt the intermolecular forces between water molecules, allowing proteins and other macromolecules to dissolve more easily. Chaotropic agents interfere with stabilizing intramolecular interactions mediated by non covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

Thermodynamics Theory of Hydrophobic Interaction Chromatography

Thermodynamic theory of HIC, directly relates to Gibb’s Free Energy equation.

The phenomenon of increased interaction, in the presence of salting out ions, is explained by a higher gain in entropy when water is transferred from the surface of a nonpolar molecule to the bulk of water.

hydrophobic interaction chromatography

In hydrophobic interaction, the entropy change is of greatest importance . If hydrophobic molecules such as aliphatic carbon chains are immersed in water, the monolayer of water molecules in contact with a carbon chain, will be in higher order than those in the bulk of the water. If two or more hydrophobic structures come together, the surface that has to be covered by, ordered water molecules decreases, some water molecules join the less ordered bulk water, and the entire system gains in entropy and thus, decreases its free energy. This state will be favourable, for energetic reasons and thus promoted.

Hydrophobic Interaction chroamtography thermodynamics

Surface Tension, van der Waals Forces Theory.

A third theory suggests that, van der Waals forces are responsible, for the hydrophobic interaction in HIC.

This is supported by the fact that, these forces should be increased as the order of water increases, in the presence of salt.

Hydrophobic Interaction Chromatography Media

In HIC, media is described based on:
  • Ligand, 
  • Ligand Density, 
  • Available Capacity.
Available Capacity is the, actual amount of protein that can bind to the media. If flow rate is included in the defined conditions then it is called as Dynamic Binding Capacity.

Dynamic Binding Capacity, is based on these factors
  • Salt Concentration, 
  • Flow Rate,
  • Temperature and 
  • pH to a lesser extent.
The Characteristics, of good Hydrophobic Interaction Chromatography Media /  Matrix are,
  • High Binding Capacity, 
  • Physical Stability, 
  • Chemical Stability and
  • The Inert Matrix.
Hydrophobic Interaction Chromatography Ligands

Ligands Used in, Hydrophobic Interaction Chromatography meida has Alkyl or Aryl Groups.

Phenyl 650 HIC matrix
Phenyl 650

butyl S 650 hydrophobic interaction matrix
Butyl S 650

Alkyl shows, pure hydrophobic character, while Aryl shows mixed behaviour.

Ligand attachment, to the matrix in HIC is done through glycidyl ether.

Binding and Elution in HIC

Binding is done at high salt concentration: 1 to 2 Molar Ammonium Sulfate or 3 Molar sodium chloride

Elution is performed, by reducing the salt concentration.

Elution of proteins from the HIC, will be in the increasing order of hydrophobicity.

  • Protein Purification Technical Resources, GE Amersham.
  • Protein Purification Technical Resources, Biorad.
  • Protein Purification Technical Resources, TOSOH Biosciences.
  • Hydrophobic Interaction Chromatography:Fundamentals and Applications in Biomedical Engineering, Andrea Mahn.
  • Hydrophobic Interaction Chromatography, Encyclopedia of Bioprocess Technology.
  • Calibration and Optimization of Hydrophobic Interaction Chromatography , Alex Olsson.