THEORY– Plant extractions for DNA is considered one of the tedious methods for high quality DNA isolations. Unlike animal tissues, which have the same tissue type in different species, plant tissues structural biomolecules and metabolites keep changing. Polysaccharides and polyphenols are two very special class of biomolecules that are very different from species to species and thus, becomes a hurdle during DNA isolation. Those biomolecules which contaminated drastically affect the manipulation of DNA isolation.
PROCEDURE-
2gm of plant sample was taken in a motor and 5ml homogenization buffer was added. Then, it was ground well for 15 minutes. Then wash it thoroughly.
15 ml of lysis buffer was added, and it was again ground well for 15 minutes.
The mixture was incubated at 65℃ for 30 minutes in microcentrifuge tubes.
The tubes were then centrifuged at 8000 rpm (rotations per minute) for 10 minutes at room temperature.
500µms supernatant was taken in microcentrifuge tube and equal volume of PCL mixture was added.
Again, tube was centrifuge at 12000 rpm for 10 minutes.
Then, the upper aqueous layer was collected (50 µL) in a new tube and chilled ethanol 200 microliter was added.
Tubes were incubated at -80℃ for 10 minutes or at 4℃ for 1hour.
Tubes were centrifuged at 12000 rpm for 12 minutes.
The supernatant was removed, pellet was air-dried and this is dissolved in µL of TE buffer.
Cells circulating in body and part of immune system are known as lymphocytes. There are mainly two main types of lymphocytes, which includes B Cells and T cells.
Peripheral Blood Lymphocyte culture:
PBLC is a technique used in Cytogenetics used for karyotyping analysis. The most convenient method to study chromosomes and by using cell culture technique it can be helpful for clinical and research purposes. The powerful mitogen for human T cell is PHA (Phytohaemagglutinin).PHA is a plant hormone, which is obtained from Red Kidney bean. The function of PHA is to bind to T Cell membrane, which stimulates metabolic activity. It is important to obtain cells at metaphase plate, since the study of Chromosome is possible and also easy to stain the chromosome at metaphase stage. During metaphase chromosomes are attached to spindle fibres.
Procedure for PBLC
Cell culturing should be done in a laminar Hood to avoid contamination.
1 ml of Venous blood should be collected
Take 7 ml of RPMI1640 ( reconstituted with 10% fetal calf serum) in sterile culture tube
Add 0.5 ml blood in culture tube
Add 0.1 ml of PHA ( Previously thawed)
Incubate this tube in sterile CO2 Incubator at 37°C FOR 69 hours
Add 40 µL of Colchicine at 69:00 hours
Incubate for 30minutes at 37°C
After Incubation centrifuge at 2000 rpm for 15 minutes
Remove supernatant and add 5 ml of Hypotonic solution
Mix gently and Incubate in waterbath for 37°C for 20 minutes
Add ice cold fixative (1:3 acetomethanol) and flush thoroughly
Centrifuge at 2000rpm for 15 minutes and remove supernatant
Repeat this above two steps for 3-4 times
Mix final suspension of 1 ml of fixative and make drop preparation in cold wet slide
Fix on flame and stain with 4% Giemsa
Observe and Scan for well spread metaphase plate in microscope
Function of each reagent
PHA: it binds to membrane of T Cell and stimulate metabolic activity and cell division
Colchicine: Suppress mitosis and cell division, but there is no interruption of chromosome multiplication
Hypotonic solution : Used for swelling of cells
Aceto-methanol: Helps in hindering cells at metaphase stage
Chromatography is a technique which is widely used for the separation of mixtures based on the physio-chemical differences between the stationary and the mobile phase of the chromatographic apparatus. The sample to be separated is dissolved in the mobile phase, which passes over the stationary phase (the immobile/fixed surface) and carries out the process of separation of molecules.
Most chromatographic processes employ and rely on these physio-chemical differences between the stationary and mobile phases for the process of separation of molecules. For example, gel permeation chromatography, ion exchange chromatography etc.
However, an exception is affinity chromatography. Unlike gel permeation and ion exchange chromatography, affinity chromatography exploits the tendency or capacity of certain biomolecules to bind specifically and non-covalently to other molecules called ligands (i.e., “bio-specificity of molecules)
PRINCIPLE:
One of the most familiar concepts for anyone involved in the studies of biochemistry is “bio-specificity”.
Specificity is a molecular recognition mechanism which operates through structural and conformational complementarity between the biomolecule and its substrate. It is known that a given enzyme only bind/react with its specific group of substrates and not with any other.
This bio-specificity may not always be limited only to enzymes. Other examples of this kind of biomolecular interactions include;
A given hormone binds only to its specific glycoprotein (known as the hormone receptor).
Antibodies specifically bind to only a given antigen which is shaped/has a confirmation specific to that antibody.
To understand this principle better with respect to affinity chromatography, let us suppose that an enzyme is to be purified from a mixture of thousands of proteins.
Let the substrate analogue (molecule resembling the substrate but not capable of reaction) specific for this enzyme (which is to be separated) be coupled to the stationary phase (let’s say the stationary phase for this particular example be e.g., agarose).
All other molecules, which have no specificity for the ligand and are incapable of binding will pass down and out of the column, (as seen in the second diagram in the picture.)
Lastly, once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength, or polarity conditions which would completely alter the strength of binding of the enzyme to the substrate and help in the elution of the desired product.
COMPLICATINS THAT ARISE DURING AFFINITY CHROMATOGRAPHY:
There are some complications which arise during affinity chromatography, they may be due to the nonspecific adsorption of sample components other than the desired one on to the matrix. Usually, when this happens, ionic and hydrophobic interactions are involved.
This complication may be taken care of by judicious choice of operating conditions (e.g., pH, temperature, or ionic strength) in such a way that the physical conditions exclusively favour the binding of the desired molecule to the substrate and not any other undesired molecule.
Another type of complication arises when one uses ligands, which interact with more than one macromolecule present in a given mixture.
IMPORTANT VARIALBES INVOLVED:
The type of matrix that is used.
The type of ligand used.
The conditions of binding and elution of the sample from the matrix.
A general discussion of these points has been given below:
Properties of the supporting matrix:
The matrix that is used in the process must be chemically inert to other molecules in order to minimize the rate of non-specific adsorption.
The matrix should possess good flow properties.
The matrix should be able to remain stable even at varying levels of pH and temperature, ionic strengths and denaturing conditions.
It should be highly porous to provide a large surface area for the attachment of ligand.
Ligand Selection:
The ligand used should be capable of forming moderately strong interactions with the desired macromolecule.
The ligand that is to be bound must possess functional groups that may be modified to form covalent linkages with the supporting matrix.
Ligand attachment:
The covalent coupling of the ligand to the supporting matrix occurs in two steps;
Activation of the matrix functional groups, and
Covalent attachment of the ligand to the matrix.
The chemical methods that are used must be relatively mild so as to ensure no/minimal damage to the ligand or the matrix.
APPLICATIONS OF AFFINITY CHROMATOGRAPHY:
Affinity chromatography has been widely used for the purification of varied number of macromolecules which are capable of showing ligand specific interactions like enzymes, hormones, antibodies, nucleic acids, membrane receptors etc.
Affinity chromatography has also been used for the purification of cells as this technique is known to affect the viability of cells less than other chromatographic techniques. Cells that have been purified using this technique include fat cells, T and B lymphocytes, spleen cells, lymph node cells etc.
An extension of affinity chromatographic principles is using magnetic gels. The gel beads contain a magnetic core that is chemically coupled to a protein ligand. The suspended cells are then allowed to interact with the microspheres. When a magnetic field is passed, the cells of interest move towards the poles of the magnets thereby getting separated. The cells can be collected by removing the magnetic field.
Immunoglobulin negative thymocytes and neuroblastoma cells are separated using this technique.
Sources:
Biophysical Chemistry principles and techniques – Upadhyay and Nath
Chromatography, broadly, is a technique to separate two phases which are mutually immiscible. The phases are brought in contact with each other and one of these phases is stationary while the other phase is a mobile phase which moves over the surface of the stationary phase or percolates it. The interactions of the sample mixture which are established after the movement of the mobile phase over the stationary phase lead to the separation of the desired compound present in the mobile phase based on the differences in the physio-chemical properties of the two mediums.
There are various techniques used in the process of chromatography like plane chromatography, paper chromatography, thin layer chromatography, column chromatography etc.
Based on these techniques, further different types of chromatographic procedures have been curated which include adsorption chromatography, partition chromatography, gel permeation chromatography, ion exchange chromatography and affinity chromatography.
GEL PERMEATION CHROMATOGRAPHY:
Gel permeation chromatography is a chromatographic procedure that separates molecules based on their molecular size. This method has various other names such as molecular sieve, gel filtration and molecular exclusion chromatography.
This method is widely used due to its many advantages, some of which are:
It is a very gentle technique that permits the separation of labile molecules.
It is a technique in which the solute recovery is almost 100%.
The reproducibility of this technique is high.
The technique is not very time consuming and is relatively inexpensive.
PRINCIPLE:
The principle of gel permeation chromatography is relatively very simple.
A long column filled with gel beads or porous glass granules is allowed to attain equilibrium with a solvent which is suitable for the separation of the desired compound or molecule.
Assuming that the mixture to be separated contains molecules of varying sizes, when this mixture will be passed through the column containing the beads;
The larger molecules pass through the interstitial spaces between the beads and do not enter the spaces inside the beads. This occurs because the diameter of the molecule is larger than the pore size of the beads.
Therefore, the larger molecules are able to pass down the column with little or no resistance.
The small molecules however, have a size which is smaller than the diameter of the pores of the beads and therefore these molecules enter the beads and reach the end of the column after a longer period of time due to the resistance that is created by the passage of the molecules through the beads.
The degree of retardation (or the extent of the time taken by the molecule to reach the bottom) is proportional to the time it spends inside the pores of the gel which is a function of the molecules pore size and the molecules size.
The molecules which have a diameter equal to or smaller than the pore size, do not enter and are said to be excluded. Therefore, the exclusion limit of a gel can be defined as the molecular weight of the smallest molecule which is not capable of entering the pores. For e.g. linear polysaccharides and fibrous proteins have a much lower exclusion limit as compared to the globular proteins of comparable molecular weight.
TYPES OF GELS USED:
A good gel filtration medium should possess a few properties or characteristics like:
The material of the gel should be chemically inert.
It should contain a small number of ionic groups.
The material of the gel should have a wide variety of particle and pore sizes.
It should have a uniform particle and pore size.
The material of the gel should be of high mechanical rigidity.
Some examples of the types of gels used in gel permeation chromatography include sephadex, polyacrylamide, agarose, styragel etc.
TECHNIQUE/PROCESS OF GEL PERMEATION CHROMATOGRAPHY:
This chromatographic technique can be performed wither by column or by thin layer chromatography techniques.
The initial step of the process is:
Preparation of the beads:
Prior to use, the gels used in the column must be converted to their swollen form by either soaking them in water or using a weak salt solution. The greater is the porosity of the gel beads, more will be the time taken for them to attain equilibrium and reach their maximum size.
If porous glass granules are used, the gel beads need not be hydrated at all.
Preparation of the column:
The gel beds, in their swollen form, are mounted or supported on a column on a glass wool plug or nylon net and the previously swollen gel is added in the form of slurry. The preparation is then allowed to settle. Air bubbles must be removed by connecting the column to a vacuum pump. It must be noted that the level of the liquid must not go lower than the top of the bed.
Addition of the sample:
The sample must be added from the top of the column using a funnel and the volume of the sample that must be added depends on the size and the type of the gel that is used in the preparation of the stationary phase.
Collection of the sample:
The eluant used is steadily added and the effluent can be collected in various various fractions in separate tubes.
Detection of sample:
The most common detection methods include the collection and analysis of the fraction and continuous methods in which the UV absorption, refractive index or radioactivity is measured.
APPLICATIONS:
Gel permeation chromatography is mainly and widely used for the separation of biomolecules and purification. Biomolecules like proteins, hormones, enzymes etc. can be separated using this technique.
This technique is especially useful for the separation of 4S and 5S tRNA.
This technique is used for the separation of salts and small molecules from macromolecules.
Gel permeation chromatography is also chiefly used for the detection of molecular weights of macromolecules.
Sources:
Biophysical Chemistry Principles and Techniques by Updhyay and Nath
Infectious diseases affect living forms on earth that include but not limited to plants, animals including human beings. Pathogenic micro organisms i.e., bacteria, virus, parasites etc attack, hinder the growth and development of an organisms and sometimes lead to death.
Infectious diseases that turn out to be pandemic have had bad effect on human beings in the history like bubonic plague, influenza, Spanish flu, avian flu and the most recent on COVID-19 (SARD-CoV-2). When the diseases spread rapidly and cross the country’s border, it is called as a pandemic.
Rapid diagnosis and treatment are the only mode of spreading the disease and to save lives. Standard traditional methods of microbial detection include – microbial culture (aerobic and anaerobic), Gram staining, colony morphology and other biochemical analysis. However, these traditional microbial methods take 5-7 days to give result, by then patients would be severely affected and sometimes might die.
Henceforth, rapid diagnosis with in a day or in hours of time is critical for effective treatment and patient management. Recently, Nucleic acid amplification technique (NAAT), a major molecular biology application has gained interest in the diagnostic field for its rapid and sensitive pathogen detection in short time. Polymerase chain reaction (PCR), a NAAT method gave hope for the early infection detection as it is fast and sensitive method in pathogen detection. PCR uses thermoresistant DNA polymerase enzyme (Taq polymerase), sequence specific primers and under specific cyclic conditions amplify the pathogenic DNA isolated from the sample to billions of copies in few hours. Due to sensitivity and speed, PCR became the choice of pathogen detection in medical microbiology field.
However, PCR has certain draw backs as follows:
Need of a thermocycler (high cost)
Need of carcinogenic material like Ethidium bromide for DNA band visualization
Need of a trained technician
Sophisticated molecular biology lab setup with at least three isolated rooms
Can’t be setup at point of care centres like rural areas
Loop-mediated Isothermal amplification (LAMP) is a revolutionary NAAT method, discovered by Notomi et al in 2000. It has the potential to rapidly detect the pathogenic DNA more sensitively and specifically in comparison with PCR at a constant temperature (isothermal). LAMP method is based on the auto cycling of the DNA using strand displacement reaction and utilizes DNA polymerase like Bst, Bsm, Gsp etc
Due to its high sensitivity, speed and efficiency, it has varied applications in medical microbiology field.
Fluorescence detection in real time. Visual detection with naked yes, gel electrophoresis or turbidity.
Fluorescence detection in real time. Visual detection is only through gel electrophoresis
Table 1: Differences between LAMP and PCR techniques
LAMP PRIMERS
LAMP makes use of 4 primers that are designed specifically to recognise 6 different regions on the target gene.
Forward inner primer (FIP) – FIP comprises of two primers namely, F2 region at the 3’ end and F1C region at 5’ end (F1C of the primer is similar to F1C portion of the target DNA).
Backward inner primer (BIP) – BIP comprises of two primers namely, B2 region at the 3’ end and B1C region at 5’ end (B1C of the primer is similar to B1C portion of the target DNA).
Forward outer primer (F3) – F3 is the outer primers and is short in its length. F3 is complementary to F3C region of the target DNA.
Backward outer primer (B3) – B3 is one of the outer primers and is complementary to B3C region on target DNA.
Loop primers – Loop forward and loop backward (LF and LB) are the two additional primers utilized in the LAMP reaction to increase the speed.
Figure 1: LAMP primers
STAGES IN THE LAMP REACTION
1. F2 portion of FIP primer binds to F2C region on the target DNA and initiates new strand synthesis and amplification.
2 F3 outer primer then binds to the F3c region on the target DNA and extends the strand by displacing the FIP associated complementary strand. This displaced strand forms a loop lie structure at the 5′ end.
3. Th synthesized ssDNA with a loop at the 5′ end assists as a template for BIP. B2 binds to B2c region on the target DNA and synthesizes new complementary strand by opening of the 5′ end loop.
4. Now, the B3 primer binds to B3c region on the target DNA and extends by displacing the BIP connected complementary strand. This results in dumbbell shaped DNA formation.
5. Then the F1 primer gets extended by opening up the loop at the 5′ end with the help of Bst DNA polymerase. At this stage, dumbbell shaped DNA become a stem loop structure and initiates the LAMP reaction. This stage is called the LAMP reaction’s second stage.
6. Further in LAMP cycling, the FIP binds to the loop region of the stem-loop DNA and initiates strand synthesis by displacing F1 primer and formation of a new loop at the 3′ end.
7. Extension happens at the 3′ end of B1 by displacing the FIP strand, forming a dumbbell shaped DNA. Self-primed DNA synthesis by strand displacement gives out one complementary strand of the original stem loop DNA along with one more stem loop DNA with a gap repair.
8. Both the new stem loop DNAs act as template for a BIP primed strand displacement reaction in the succeeding cycles. Consequently, for every half LAMP cycle, 13 fold amplification of the target DNA occurs.
The ultimate amplification LAMP products contain combination of stem loop DNA with varied lengths looking like a cauliflower like structure with multiple loops.
LAMP REACTION SET UP
To setup LAMP assay, target DNA, an isothermal DNA polymerase with strand displacement activity, primers and buffer are sufficient. LAMP assay can be setup in a simple water bath or a heat block at a constant temperature (ideally at 65°C).
Figure 2: Over view of LAMP reaction
LAMP DETECTION
LAMP reaction can be detected as follows:
Fluorescence based detection: using florescent dyes like SYBR green, Pico green, Eva green
Figure 3: LAMP detection using SYBR green fluorescent dye
Visual detection: Based on turbidity and precipitation in the positively reacted tubes. Leuco crystal violet is a dye that detects positive reaction based on turbidity.
Figure 4: LAMP detection based on turbidometry
Colorimetric dyes are also used as they react with the free Mg2+ being produced in the reaction. Colour change happens based on the pH change. For example, phenol red show pink colour before the reaction and turns yellow if the sample is positive for the pathogen (as the pathogenic DNA multiplies, more Mg2+ will be released into the reaction tube).
Figure 5: Colorimetric detection of LAMP reaction (Source: NEB)
ADVANTAGES OF LAMP TECHNIQUE
Require simple water bath or heat block (no costly thermocycler).
Amplification at isothermal conditions.
More specificity and sensitivity as it utilizes 4-6 primers.
Cost effective
Easy deployment at point of care centres at rural areas.
No trained technician is required. Setup is quite simple.
APPLICATIONS OF LAMP TECHNIQUE
Rapid diagnosis of bacterial, viral, fungal and parasitic organisms.
Helps to detect pathogens at both genus level and species level. In case of viruses, various strains can be easily differentiated.
Centrifugation is one of the most extensively used technique in research and development fields of biochemistry, molecular biology, biochemistry and pharmaceutical industries for varied applications like isolation of cells, fractionation of sub cellular particles and other macromolecules for analytical and clinical applications.
Definition
Centrifugation is a process of separation (or concentration) of particles from suspended medium based on their size, shape, density, viscosity of the medium, rotor speed etc. Centrifugal force is a key in this technique to separate the particles in less time and it acts against gravitational force. Figure 1 gives overview of centrifugation process from muscle tissue.
Figure 1: Centrifugation overview
Principle:
In general, when a liquid suspension is placed idle for some time, particles of bigger size/density will start to settle at the bottom of the container because of gravitational force and so on. But, this is a slow process and can’t be applied practically. Centrifugation works on centrifugal force to separate the particles in a suspension in less time with more efficiency.
When a body with mass ‘m’ is rotating in a circle with radius ‘r’ and velocity ‘v’, the force acting on the body is measured using the following formula 1.
F = mv2/r
Where,
F = centrifugal force,
m = mass of body,
v = velocity of the body,
r = radius of circle of rotation.
The gravitational force acting on the body ‘m’ is calculated using the formula 2: G = mg
Where,
G = gravitational force
g = acceleration due to gravity
U
The centrifugal force is further calculated using the formulae 1 and 2 as follows:
C = F/G = mv2/mgr = v2/gr
Since, v = 2π r n
Where,
n = speed of rotation
C = F/G = (2π r n) 2/g r = 4π2 r2n2 = 2π2/g D n2 = kD n2
where,
k = 2π2/g = constant
D = maximum diameter of the centrifuge
D is able to measured either from centrifuge center to the free surface of the liquid or to the tip of the centrifuge tube.
From the equation C = kDn2 it was evident that,
Centrifugal effect ∝ diameter of centrifuge
Centrifugal effect ∝ (speed of rotation)2.
When a liquid suspension containing container is rotated at a certain speed called revolutions per minute (RPM), particles will move at a certain speed away from the axis of rotation. The force that’s being generated on the particles to move away from the centre is called relative centrifugal force (RCF). RCF depends mainly on the rotational speed (measured in RPM) and the distance of the particles from the centre of the rotation (rotor).
RCF = 11.2 × r (RPM/1000)2
Where,
r – Distance in centimeters
More the density of the particles, faster is the settlement at the bottom of the tube, while less dense particles will be floating in the liquid. The rate of sedimentation depends on the size and density of the particles and can be explained by Stokes equation (explains movement of a sphere in a gravitational field).
Where,
V = viscosity of the medium
d = diameter of the sphere
p = particle density
L = medium density
n = viscosity of medium
g = gravitational force
Stokes equation explains behavior of particles based on the rate of particle sedimentation as follows:
directly proportional to the size of the particle
directly proportional to the difference between the particle and the medium densities
zero when the particles and medium exhibits same density values
decreases when the medium viscosity increases
increases as the gravitational force increases
Table 1: Densities of cells and sub cellular fractions
The particles that gets settled at the bottom forms ‘pellet’ while the liquid suspension with lighter particles or no particles is called ‘supernatant’. Therefore, centrifugation is a process that utilizes centrifugal force for the sedimentation of particles.
Figure 2: Densities and sedimentation coefficients of biomolecules, cell organelles and viruses
For example, ‘m’ is a particle in a centrifuge tube suspended in a liquid. During centrifugation process, the particle is influenced by three kinds of forces: FC– the centrifugal force, FB – the buoyant force and Ff – the frictional force between the particle and the liquid.
Figure 3: Centrifugal force
Centrifuge
Centrifuge is a tool designed to separate particles in the liquid suspension based on the centrifugation principle. It is operated using an electric motor that enables an object to move around in a fixed axis when a perpendicular force is applied to the axis.
Figure 4a: Front view of a typical centrifuge
Figure 4b: Rear view of a typical centrifuge
Centrifuge comprises of three major components:
Rotor – Holds containers (tubes/bottles etc) containing liquid suspension to be centrifuged. Rotors of different types and sizes are available
Fixedangle rotor – Requires short time to sediment particles as they need to travel only a little distance.
Figure 5a: Fixed angle rotor
Swinging bucket rotor – Allows better separation of particles as the particles have to move long distance. Stronger pellet is formed and supernatant can be easily removed.
Figure 5b: Swinging bucket rotor
Drive shaft – Helps hold rotors which in turn connect to motor.
Motor – Helps to rotate the rotor based on the input speed by providing power.
All the major centrifuge components are surrounded by a protective cabinet with operating controls and indicator dials for speed and time mounted on it. Centrifuges come with brake system to control rotor and allow it to come to standstill when the centrifugation run gets completed. Refrigerated centrifuges allow option to control temperature so that the delicate biological samples won’t be degenerated during the process.
Types of centrifugation techniques
There are two major types of centrifugation techniques to separate particles.
Differential centrifugation
Density gradient centrifugation
Isopycnic centrifugation
Rate-zonal centrifugation
Differential centrifugation:
This is the simplest type of particle separation form, also called ‘pelleting’ down the particles. Sedimentation occurs at different rates based on the density of the particles. More dense particles will sediment fast and the lighter particles will be floating in the suspension. Sedimentation of the particles also depends on the centrifugal force applied. As the centrifugal force increased, pellet with decreased sedimentation rate will be formed and vice versa.
Differential centrifugation is applied during cell harvest or sub cellular fractionation from a tissue homogenate. When lower centrifugal force is applied, dense particles like nuclei, membrane vesicles etc gets pelleted first. To further pellet next order particles like mitochondria etc, more centrifugal force is applied. Greater than or equal to four differential centrifugation cycles are applied for sub cellular fractionation of the tissue homogenate. However, this process faces carry over contamination of the particles from previous fraction and not purity is less.
Figure 6: Differential centrifugation process overview
Density gradient centrifugation: This method is mainly applicable to separate sub cellular particles and other macromolecules with more purity.
In this process, gradient media of different density are layered one above the other, more dense at the bottom of the tube and the lightest at the top. The cell fractionate that need to be separated is placed on the top of the gradient layer and the centrifugal force is applied.
Figure 7: Density gradient process overview
Density gradient method is further classified into two types as follows:
Isopycnic centrifugation:
This process is also known as buoyant or equilibrium separation. In this process, particles are separated base on density. Particle size plays a role when the density of the particles and the surrounding medium is same. When the centrifugal force is applied, initially the sample and gradient gets mix uniformly and the particles move through the gradient until the density of the particles and gradient medium becomes same. Now, the gradient is called as ‘isopycnic’ and the particles get separated based on their buoyancy. Therefore, it is important to make sure that the gradient medium is always dense than the particles to be separated. Particles get separated in the gradient medium in different layers, but never settle to the bottom of the tube. Gradient medium varies depending upon the kind of material being separated. Continuous gradient method is good for analytical separation while discontinuous gradient is more suitable for biological applications (E.g.: separation of lymphocytes from blood).
Table 2: Common density gradient media used for isopycnic centrifugation process
Rate zonal centrifugation:
The carryover contamination of particles in differential centrifugation is prevented by implementing rate zonal centrifugation. In this process, sample was layered in a narrow zone on the top of the density gradient and the centrifugal force is applied. Particles segregate based on their size and mass rather than density, and also on the centrifugal force. As a result, narrow load zone prevents less sample volume (≤10%) that can be accommodated on the density gradient and it further stabilizes the bands and allows medium of increasing density and viscosity. Centrifugation is applied for a short time at a low speed. As the density of the particles is more than the density of the gradient, there is a chance that all the particles form pellet if the centrifugation continues for a long time.
Figure 8: Rate zonal and isopycnic centrifugation processes overview
Common applications of centrifugation
Production of drugs and other biological products
Separation of subcellular particles
Separation of blood and urine components in forensic analysis
Ion exchange chromatography is based on the principle of reversible exchange of ions and polar molecules by retaining the sample molecules on a column (inert support medium).
It is based on electrostatic force of attraction or ionic interactions between the ions and the surface of the stationary phase which has ionic functional groups (R-X)
Ion exchange chromatography is carried out in columns packed in an ion exchanger, which is an inert insoluble support medium. Ions bound electrostatically to the column are known as counterions.
The stationary phase can be ion exchange resins that carry charged functional groups to interact with oppositely charged groups in the sample.
It can be employed on charged molecules like ions, amino acids, small nucleotides, large proteins, etc.
The sample containing the ionic species which has to be separated is allowed to percolate through the exchanger for a sufficient amount of time so that equilibrium can be achieved.
Types of exchanges in ion exchange chromatography
Based on charge:
Anion-exchange chromatography:
This uses ion exchange resins containing positively charged groups like diethyl-aminoethyl groups. In solution, resins are coated with positively charged counter ions, which have an affinity for molecules with net negative charges on the surface. Also known as “Basic ion exchange” materials.
Cation-exchange chromatography
Cation exchange chromatography is a technique that uses a negatively charged ion exchange resin which has an affinity for molecules having net positive charges on their surface. Also known as “Acidic ion exchange” materials.
The total number of equivalents of replaceable protons per unit volume of resin determines the exchange capacity of the resin.
Based on this there are two kinds of exchangers:
Strong exchangers
Strong ion exchangers show no variation in ion exchange capacity with changes in pH. They are prepared with a tertiary amine, yielding a strongly basic quaternary ammonium group.
Weak exchangers
They are ionized over only a limited pH range. Weak anion exchanger is prepared with secondary amines which yield a weakly basic tertiary amine.
What is the Isoelectric point in ion exchange chromatography?
Ion exchange chromatography is based on the different charges of ions and the electrostatic force between the ionic charges and that of the column of chromatography.
Isoelectric point is the pH at which the overall number of negative and positive charges is equal to zero. Thus, no ion exchange takes place at the isoelectric point.
Resins used in ion exchange chromatography:
Applications of ion exchange chromatography
Used in in amino acid analysis. Amino acids are known as “autoanalyzer” and this is based on ion exchange principle
Ion exchange has also been extensively used to determine the base composition of nucleic acids. Treatment with DNAses and RNAses which results in a mixture of nucleotides can be readily separated by ion exchange chromatography.
In Biological applications, ultrapure, metal ion free reagents are needed. This is commercially performed by ion exchange chromatography.
Ion exchange chromatography has been used for the separations of many vitamins, other biological amines, and organic acids and bases.
Mutagenic potential of any compound can be assessed by using AMES test. This test can be also called as test for mutagenicity (refers to compounds or substances causing mutations).
Whether a given chemical can cause mutations in the DNA of test organism or not bacteria are utilized in this test.
To determine whether the chemical at hand is a mutagen or not, this test was developed in the year 1970 by Bruce N Ames.
Examples of mutagens: certain chemicals like Ethidium bromide, which is a carcinogenic agent, UV radiations and X-rays can be called as potential mutagens.
If the testing substance yields positive result, then it indicates that the substance is a potential carcinogen or mutagen.
Objective of the test:
This test is specifically employed to test mutagenic activity of chemicals.
The test is employed in sample bacteria to study mutations which it confers and to identify the potential mutagen and its pattern of causing mutations.
This test is also utilized in testing plumbing products in contact with drinking water, using the reversion system of Salmonella typhimurium histidine (His).
Principle of Ames test:
Strains of bacteria carrying a particular mutation are used in Ames test. Strains of bacteria like E.coli, salmonella are used.
Histidine or tryptophan operon of salmonella or E.coili is induced with point mutations (change in single base) which make the bacteria deprived of producing the corresponding amino acid.
Organisms grow only when histidine or tryptophanis supplied, and this is because of the induced point mutations.
Media containing certain chemicals is used to culture His-salmonella which results in mutation in histidine encoding gene, making them able to regain the capacity to synthesize histidine. (His+).
This can be explained as, when base substitution or frame shifts (shift in the reading frame) occurs within the gene, it causes reversion to amino acid prototrophy due to reverse mutations.
The bacteria obtained from the reverted culture will then be able to grow in histidine- or tryptophan- deficient media.
By exposing amino acid requiring organisms to different chemical concentrations and selecting for reversion event, samples mutagenic potential can be thoroughly assessed.
For selection purpose media lacking the specific amino acids (histidine and tryptophan) are used to allow growth of only those cells that have undergone prototrophy reversion to histidine and tryptophan to grow and survive.
If the reversion is seen in the test sample used, it indicated that the substance used for testing is a mutagen.
Procedure:
Isolation of auxotrophic strain (strain which lacks ability to synthesize an essential compound or nutrient) from Salmonella for histidine. (His-).
Test suspension is prepared with histidine negative (His-) Salmonella in a plain buffer along with the test chemical to be used. Small amount of histidine is also required to be added to the mixture. (histidine added in smaller amounts helps in the growth of bacteria. As histidine is applied in smaller amounts, once histidine (his) depletes only those bacteria mutated to gain the ability to synthesize histidine will form the colonies).
Control isprepared with his- salmonella without adding test chemicals.
Suspensions are incubated at temperatures of 37 degrees, which is the temperature set inside incubator for 20 minutes.
After 20min, the suspension is taken and is spread on the agar plates. (Perform this under laminar hood.)
Colonies can be observed after 48 hours. Count the number of colonies in each of the agar plates.
Interpretation of results:
Chemical mutagenicity is proportional to number of colonies observed.
Comparing to control, if there are larger number of colonies, then result can be interpreted as, the chemicals used for testing are potential mutagens.
If less number of colonies is observed, probably this could be due to point mutations which occurred spontaneously on histidine encoding gene.
Applications:
Screens chemicals that are potential carcinogens or mutagens. Example- AF-2 which is a food additive commonly known as Furylfuramide. This was used as food additives but later was withdrawn in the year 1974 from the market after been identified as mutagenic to bacteria in-vitro.
Ames test has the ability to detect mutants from a larger population of bacteria with higher sensitivity.
This test is commonly known as test for mutagenicity and not as Carcinogenicity but most of the mutagens, almost above 90% detected by Ames test are known to cause cancer.
The defective gene of bacteria can be mutated into functional gene, as Ames is a bacterial reverse mutation assay.
Several samples like dye, drugs, reagents, cosmetics are been tested using Ames test to detect mutagens present in them.
Merits:
Low cost affordable assay.
The assay is simple, rapid and less time consuming.
Can detect mutants with high sensitivity even from a very large population.
Limitations:
Some Cancer causing substances in laboratory animals does not give positive result for Ames test. Example- Dioxins, highly toxic chemical compounds.
This assay cannot be a perfect model for humans, as strains used are of Salmonella Typhimurium.
In molecular biology the isolation and purification of DNA from cell is most important process.
The bacterial cell that is used in this procedure should be grown in suitable media under favourable conditions and harvested in late log to early stationary phase.
Along with DNA bacterial cell contain RNA, lipids, protein which needs to separate.
The first step in DNA isolation is to disrupt the cell membrane and this is done by SDS (Sodium Dodecyl Sulphate)
Endogenous nucleases present on human fingertips can degrade the nucleic acid during purification. This degradation by nucleases can be prevented by chelating mg2+ ions using EDTA.
Mg2+ ion is a necessary cofactor for the action of the nucleases.
Proteinase K is used to degrade protein in the disrupted cell.
And the function of phenol and chloroform is to denature and separate the protein from DNA.
The denatured protein makes a layer between aqueous and the organic phase.
DNA from the disrupted cell is precipitated by cold absolute ethanol or isopropanol.
Requirements:
LB media
E.coli DH5α cells
TE buffer
10% SDS
Proteinase K
Phenol:Chloroform (1:1)
5M Sodium acetate
Isopropanol
70%ethanol
Autoclaved distilled water
Eppendorf tube
Micropipette
Microtips
Micro-centrifuge
Procedure:
Take 1.5ml of the bacterial culture (grown overnight) and harvest it by using centrifuge at 5000 rpm for 3-4 minutes.
Discard the supernatant and add 600μl lysis buffer and incubate at room temperature for 10 minutes.
Then add 200μl 10% SDS and 5μl proteinase K to the cells and incubate for 10-15 at 37o C.
Now incubate the tubes in water bath for 10 minutes at 60o C and immediately transfer to ice bucket and keep for 5 minutes.
Adding 500μl phenol: chloroform solution in 1:1 ratio and mix by inversion for 1-2 minutes, incubate for 5 minutes.
After incubation centrifuge the mix at 10000 rpm for 10 minutes at 4o C.
Now carefully transfer the upper aqueous layer to the fresh tube.
Add 100μl 5M sodium acetate and equal amount of isopropanol mix by inversion and store at-20o C for 1 hour or overnight.
Centrifuge the mix at 5000 rpm for 10 minutes and discard the supernatant.
Then add 1 ml of 70% cold ethanol and mix by inversion.
Again centrifuge at 5000 rpm for 10 minutes and discard supernatant.
Air dry the pellet and add 40μl TE buffer and store at 4o C for further use.
Precaution:
To avoid mechanical disruption of DNA cut tips should be used.
The incubation period of proteinase K depends on the source of DNA and should be extended.
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