Unit

13

Methods in Biology

Unit Map
13.A Molecular biology and recombinant DNA methods 720
13.B Histochemical and immunotechniques 749
13.C Biophysical methods 753
13.D Statistical Methods 768
13.E Radiolabeling techniques 785
13.F Microscopic techniques 790
13.G Electrophysiological methods 796
Practice MCQs 801

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 Methods in Biology 720

Molecular biology and recombinant DNA methods

13.A.1 Isolation and purification of RNA and DNA

Isolation and purification of nucleic acids are very important for any molecular biology experiment. It is the starting material
for all types of molecular biology experiments like restriction digestion, cloning, PCR, DNA-libraries and sequencing. By
manipulating this extracted DNA or RNA and new DNA with different characteristics can be obtained. Genetic engineering
process relies on DNA, RNA and proteins only. The basic process of extracting DNA involves the release of DNA from the
cells, purification of the DNA to be used in the experiments. The pH of all solutions is maintained at pH-8.0 throughout the
extraction procedure.
The isolation and purification of DNA mainly include four steps
i. Cell lysis
ii. Enzymatic treatment
iii. Purification of DNA/RNA
iv. Quantification of DNA or RNA

i. Cells lysis Cell lysis is needed in order to release their components from the cells. The nature of the treatment will vary
widely according to the cell type. For cell lysis we need a culture of bacteria or yeast cells of extracting DNA which is
homogenized so that individual cells can be lysed. The cell walls present in bacteria which are needed to be lysed to release
the cell content is carried out by using lysozyme. The other material which can be used is EDTA and SDS detergent. EDTA
acts as chelating agent, and chelates divalent cations which act as co-factor for enzymes. SDS an anionic detergents break
the di-sulphide binds and helps the membrane proteins to degrade. In case of plants the cell walls are degraded either by
enzymatic treatment like use of pectinase, cellulose etc. The alternate method of breaking the cell wall of plants and fungi is
by using liquid N2 (liquid Nitrogen) and crushing/homogenizing the cells in mortar-pestle. Further cell lysis is carried out by
treatment with mild anionic detergent CTAB (Cetyl Trimethyl Amino Bromine). - mercaptoethanol can be used to break
the thio bonds.
ii. Enzymatic treatment Removal of RNA from a mixture of nucleic acids is easily achieved by treatment with RNase which
is a very heat stable enzyme. Protein contamination can be removed by digestion with a proteolytic enzyme such as
proteinase K.
iii. Purification of nucleic acids
 Phenol chloroform extraction To get pure DNA solution we need to remove proteins and other impurities. This is
achieved by extraction with phenol or a mixture of phenol and chloroform. Phenol and chloroform are immiscible with
water. When the mixture is vigorously agitated, the proteins will be denatured; lipids and carbohydrates solubilize in the
organic solvent and get precipitated at the interphase, when centrifugal at high-speed (15000 rpm). The aqueous phase
containing the soluble DNA for the top layer and the solvent forms the bottom phase.
 Alcohol precipitation After phenol extraction the sample which is obtained by above extraction will be free from
protein impurities and will be much diluted so to concentrate the sample alcohol precipitation is done by precipitating
nucleic acids using chilled ethanol or isopropanol. A small vol (1/10) of 3 M Sodium Acetate solutions is added before
precipitation step. The acetate ions help form the network and thus enhance precipitation. On addition of alcohol a
precipitate will appear which can then be collected at the bottom of the test tube by centrifugation.
 Centrifugation As mentioned in above paragraph centrifugation is used in DNA purification for separation of cellular
debris from solution and for recovering precipitated nucleic acids. Other method which can be used is cesium gradient
centrifugation for separation of plasmid DNA from bacterial genomic DNA, or of RNA from DNA. Sucrose gradient
can also be used for size selection of large DNA fragments when constructing genomic library.
 Plasmid Extraction Plasmids are extracted by alkali denaturation method in which, bacterial cells with the desired
plasmid are lysed under alkaline conditions and the crude lysate is purified using centrifugation. Three buffer solutions
are mainly used suspension buffer, neutralization buffer and lysis buffer. Suspension buffer contains glucose, EDTA
and Tris HCl buffer. Glucose maintains osmotic pressure and EDTA chelates divalent ions like Mg2+, Ca2+ which
prevents DNases from damaging the plasmid and also helps in destabilizing the cell wall. Lysis buffer contains NaOH
and SDS. NaOH helps to break cell wall and disrupts hydrogen bonding between DNA bases converting dsDNA
including genomic DNA and plasmid DNA into ssDNA. SDS solublizes the phospholipids and protein component of
bacterial cell membrane leading to lysis and release of cell content to obtain plasma. Neutralization buffer contains
potassium acetate, glacial acetic acid and water. Potassium acetate decreases alkalinity of mixture. The potassium
acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a

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721 Methods in Biology

partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution. Acetic
acid neutralizes the pH, allowing the DNA strands to renature.
 Column purification Mainly two types of chromatography can be done for purification of nucleic acids like size
exclusion chromatography and affinity chromatography. In size exclusion chromatography sample mix is passed
through a matrix of small porous beads. Smaller molecules such as salts and some nucleotides will enter the beads
whereas larger molecules such as longer nucleic acid will pass right through the column. In affinity chromatography the
macromolecule will bind to resins of column. The resins could be an anionic resins or oligo-dT sequences which
specifically bind to the poly-A tail of eukaryotic mRNA molecules. In both chromatography cases undesirable
molecules can be washed from the column and elution of nucleic acid bound to column can be eluted out by changing
pH conditions or other.
 Quantitation by spectrophotometer: Both quality and quantity of DNA

13.A.2 Different separation methods

Chromatography It is an analytical process for the separation of mixture which involves passing of a sample (analyte solute)
in the mobile phase; often in a stream of solvent, through the stationary phase. Some form of material that will provide
resistance by virtue of chemical interaction between components of sample and the material.
Chromatography is defined as a separation technique based on partition or distribution of a sample (solute) between a mobile
phase and a stationary phase.

Stationary phase May be a solid, gel, liquid or a solid / liquid mixture immobilized.
Mobile Phase May be liquid / gaseous for which is passed over the stationary phase after the mixture of analytes to be
separated has been applied to the stationary phase.
The basis of all the chromatography in the distribution or Kd (Patton cost) and describes the way in and the compound
distributes between two immiscible phase.
For such two phase A and B Kd is

Kα2
Conc. in phase A Conc. in phase B

It is widely used because of following advantages

 Very small quantities of substances can be analyzed quantitatively and qualitatively. Equipment is very simple.
 Actual operation is fairly simple and no special skill is required.
 The results are remarkable reproducible.

The basic technique

 Stationary Phase absorbent.
 Mobile Phase Gas/liquid moves the sample through a region containing solid/liquid.
 The molecular species separate as they differ in their distribution between two phases and it depends on driving force and
retarding force.

 HPLC High Performance Length Chromatography

It is a chromatographic technique that can separate a mixture of compounds and identify, quantify and purify the individual
components of mixture.
 Principle Organic solvents are used as the mobile phase. The analyte is first dissolved in a solvent and then force it
through a chromatographic column under high pressure. In the column, mixture is resolved into its components. The
stationary phase is defined as the immobile packing material in the column.
As the analyte is forced under high pressure, it spends less time in the column, leading to narrower phase and hence
better selectivity and better sensitivity. Often, a gradient, over time in the solvent composition passing through the
column is used to separate analyte mixtures.
 Stationary Phase Three forms are available, based on a rigid solid as opposed to gel, structure as the material need to
withstand high pressures generated in the column. All forms are spherical particles of a uniform size to minimize spare
for diffusion and hence brand broadening to occur.
Three forms are
i. Microporous ramify through the particles and are generally 5-10 nm in diameter.
ii. Pellicular In which porous particles are coated onto an inert solid concentration such as a glass bead of bout 40mm
in diameter.
iii. Bonded In which stationary phase is chemically bonded onto on inert support (Silica).

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 Methods in Biology 722

 Mobile Phase The choice of mobile phase depends on the separation being carried out.
Some common properties are
 Purity Very high purity solvents with no particulate matter. Suspended particles tend to plug the column and
chemical impurities produce sporous peak in detector. To purify laser grade solvent multiple filter is used.
 Low Viscosity High viscous solution are avoided as they require longer time to pass through the column which
results in peak broadening, power resolution and higher pressure to force solvent through the column.
 Chemical Inertness Mobile phase must not react with any sample.
 Degassing It should be degassed to avoid air bubbles in column or detector and partially block the column and is
done by warming, storing, vacuum ultrasonication.

 Ion Exchange chromatography (IEC)

 Principle It relies on the attraction between opposite charged particles. Many biological materials like amino acids and
proteins have ionisable groups that may carry a net positive or net negative charge. IEC is frequently chosen for
separation and purification of proteins, peptides, nucleic acids, polynucleotide and other charged molecules mainly
because of high resolving power and high capacity.
Two types of Ion exchanges
i. Cation Passes negatively charged groups and attracts positively charged cation. It is also called acidic ion exchanges
because their negative charges result from ionisation of acidic groups.
ii. Anion has positively charge groups and attracts negatively charged anions. It is also called as basic ion exchangers
as negative charges results from association of protons with basic groups.
Ion exchange much consists of five main steps
i. Diffusion of ion to the exchanger surface This occurs very quickly in homogenous solutions.
ii. Diffusion of ion through the matrix structure of the exchanger to the exchanger site It is dependent on degree of
cross linkage of exchanger and concentration of soln. This process is thought to be a feature that controls the rate of
whole ion exchange process.
iii. Exchange of ions at the exchange site It occurs instantaneously and is an equilibrium process.
Cation exchanger
RSO3- --- Na+ + N+ H3 R1 ↔ RSO3--- N+ H3 R1 + Na+
Bound ion Exchanged ion

Anion Exchanger
(R)4 N+ ---- Cl- + -OOCR’ ↔ (R)4 N+ ---- -OOCR’ + Cl-

The more highly charged the ionized molecules to be exchanged, lighter it binds to the exchanger and the less readily
it is displayed by other ions.
iv. Diffusion of the exchanged ion through the exchanger to the surface.
v. Selective adsorption by the eluent and diffusion of the molecule into external eluent Selective desorption of the
bound ion is achieved by changes in pH and/or ionic concentration or by affinity elution.

 Material Matrices used include polystyrene, cellulose and agarose. Final ionic groups include SO 3- and –N+R3. Ion
exchangers are stable upto 600C and separation is carried out within these temps because very high temp decreases
viscosity of mobile phase and thereby increasing efficiency of separation.
 Applications Ion exchange chromatography is prominently used as preparatory chromatography to isolate a desired
compound from mixture. Hence the applications are meant to obtain pure compounds.
 For deionisation and softening of water.
 Purification of solution to keep them ion free.
 In biochemistry for separation of drugs and metabolites from blood, urine etc.
 Separation of organic mixture of acidic and basic compounds.
 For extraction of enzymes from tissues.
 Uses
Ion exchange chromatography has many uses including
 Separation of proteins from foods, for example, to investigate the effects of individual food components on health
this type of analysis is used in nutrition research.
 Separation of high value proteins from substances.

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723 Methods in Biology

 Limitations
 Costly equipment and more expensive chemicals
 Turbidity should be below 10ppm.

 Affinity chromatography
It does not rely on difference in physical properties of molecules to be separated. It was originally developed for purification
of enzymes, but exceeded for nucleic acids, nucleotides, immunoglobins, and membrane receptors and even to whole cells
and cell fragments. This technique requires that the material to be isolated is capable of binding reversibly to a specific
ligand that is attached to an insoluble matrix.

K1
M + L ↔ ML
K-1
Macromolecule Ligand complex

When a complex mixture containing the specific compound to be purified is added to immobilize ligand, generally
contained in a conventional chromatography column, only that compound will bind to the ligand. All other compounds can
then be washed away and the compound is subsequently recovered from the ligand.
It requires preliminary knowledge of the structure and biological specificity of the compound to be purified so that
separation conditions can be planned.
In case of enzyme a ligand can be S, IRI, RI or allosteric activator.

Matrices used
Cross linked dextrans, agarose, polyacrylamide, silica, porous glass, cellulose, and polystyrene.

Applications
 Many enzymes and other protein including receptor proteins and Igs have been purified by this technique.
 It can be used for nucleic acids also. m-RNA is isolated by selective hybrid poly (u)- sepharose 48 by exploiting its Poly
A tail.
 Single stranded DNA can be used for isolating complementary DNA or RNA. It is performed by using SS DNA on nitro
cellulose membrane.
 It is used for separation of mixture of cells into homogenous populations.
Uses
 It is specific and high degree of purity is obtained.
 The absorbed material is separated from proteolytic enzyme and can be stabilized by ligand binding at active site.
 Adsorbent can be regenerated several times.
Limitation
 High cost
 Difficulties associated with scale up and high labour intensity

 Paper Chromatography
Principle The principle involved is partition chromatography where in the substances are distributed or partitioned between
to liquid phases. One phase is the water which is held in pores of filter paper used and other phase is that of mobile phase
which moves over the paper. The compounds in the mixture get separated due to differences in their affinity towards water
(in stationary phase) and mobile phase solvents during the movement of mobile phase under the capillary action of pores in
the paper.
The principle can also be adsorption chromatography between solid and liquid phases, where in the stationary phase is the
solid surface of paper and the liquid phase is of mobile phase. But most of the applications of paper chromatography work
on the principle of partition chromatography i.e. partitioned between to liquid phases.

Material used
Chromatography paper (stationary phase) Coffee filter or blotting paper or paper towel may also work. Different papers
have different properties and will separate substances more or less successfully. Chromatography chamber. 1 liter glass jar
 Paper clip.
 Skewer (or pencil).

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