1
Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
ADVANCED TECHNIQUES IN FIELD STUDIES: GENERAL CONCEPTS AND APPLICATIONS
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Extraction of DNA samples
PCR
RTPCR
DNA Sequencing
DNA fingerprinting
Southern blotting and its applications
EXTRACTION OF DNA SAMPLES
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DNA is the blueprint for life and almost every living thing contains DNA. DNA isolation
(extraction) is one of the most basic and essential techniques in the study of DNA. , DNA
Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in which
it normally resides. The extraction of DNA from cells and its purification are of primary
importance to the field of biotechnology, it is the first steps in the analysis and manipulation
of DNA and it allows scientists to detect genetic disorders, produce DNA fingerprints of
individuals etc.
DNA can be extracted from many types of cells.
The first step is to lyse or break open the cell:
Break open the cells or virus containing the DNA of interest is commonly referred to as cell
disruption or cell lysis. This is done to expose the DNA within.
Cell lysis is commonly achieved by chemical or physical methods such as
blending, sonicating, bead beating the sample or grinding a piece of tissue in a blender.
Removing membrane lipids by adding a detergent or surfactants also helps in cell lysis.
DNA associated proteins, as well as other cellular proteins, may be degraded with the
addition of a protease. Precipitation of the protein is aided by the addition of a salt such as
ammonium or sodium acetate sometimes a salt solution such as NaCl and a detergent
solution containing the compound SDS (sodiumdodecyl sulfate) is added.
Vortexing with phenol (sometimes heated) is often effective for breaking down protienacious
cellular walls or viral capsids.
The addition of a detergent such as SDS is often necessary to remove lipid membranes.
Removing RNA by adding an RNase (almost always done).
These solutions break down and emulsify the fat & proteins that make up a cell membrane.
The next step is DNA purification from detergents, proteins, salts and reagents used during
cell lysis step.
The most commonly used DNA purification procedures are:
Ethanol precipitation - usually by ice-cold ethanol or isopropanol.
Ethanol is added because DNA is soluble in water.
Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon
centrifugation.
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That is the alcohol causes DNA to precipitate, or settle out of the solution, leaving behind all
the cellular components that aren't soluble in alcohol.
The DNA is insoluble in the alcohol and will come out of solution, and the alcohol serves as
a wash to remove the salt previously added.
The resultant DNA pellet is washed with cold alcohol again and centrifuge for retrieval of the
pellet.
After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer
such as Tris or TE.
Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium
acetate.
Phenol-Chloroform extraction in which phenol denatures proteins in the sample.
After centrifugation of the sample, denaturated proteins stay in organic phase while aqueous
phase containing nucleic acid is mixed with the chloroform that removes phenol residues
from solution. (Note: for DNA isolation in used phenol buffered to pH 8, RNA must be
isolated using acidic phenol.)
When the sample is vortexed with phenol-chloroform and centrifuged the proteins will
remain in the organic phase and can be drawn off carefully.
The DNA will be found at the interface between the two phases.
Minicolumv purification that relies on the fact that the nucleic acid may bind (adsorption) to
the solid phase (silica or other) depending on the pH and the salt content of the buffer.
Finally after DNA purification the DNA can be spooled (wound) on a stirring rod and pulled
from the solution at this point.
Presence of DNA can be confirmed by electrophoresing on an agarose gel containing
ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under
UV light.
Genomic DNA extraction: Extraction of DNA basically consists of four major steps:
Preparation of a cell extract: To extract DNA from a tissue/cells of interest, the cells have
to be separated and the cell membranes have to be disrupted. The "Extraction buffer" helps in
carrying out these processes. Chemicals such as EDTA (Ethylene Diamine Tetra Acetate)
which removes Mg2+ ions that are essential for preserving the overall structure of the cell
membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes
by removing the lipids of the cell membranes are included in the extraction buffer. Having
lysed the cells, the final step in the preparation of a cell extract is removal of insoluble cell
debris. Cell debris and partially digested organelles etc. can be pelleted by centrifugation
leaving the cell extract as a reasonably clear supernatant.
Purification of DNA from cell extract: In addition to DNA the cell extract will contain
significant quantities of protein and RNA. A variety of procedures can be used to remove
these contaminants, leaving the DNA in a pure form. The standard way to de-proteinize a cell
extract is to add phenol or a 1:1 mixture of phenol:chloroform. These organic solvents
precipitate proteins but leave the nucleic acids in aqueous solutions. The aqueous solution of
nucleic acid can be removed with a pipette. The effective way to remove RNA is with the
enzyme ribonuclease, which will rapidly degrade these molecules into ribonucleotide
subunits.
Concentration of DNA samples: The most frequently used method of concentration is
ethanol precipitation. In the presence of salt and at a temperature of -20 °C or less, absolute
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ethanol will efficiently precipitate polymeric nucleic acids. With a concentrated solution of
DNA one can use a glass rod to pull out the adhering DNA strands while for dilute solutions
precipitated DNA can be collected by centrifugation and re-dissolving in an appropriate
volume of water.
Measurement of purity and DNA concentration: DNA concentrations can be accurately
measured by UV absorbance spectrometry. The amount of UV radiation absorbed by a
solution of DNA is directly proportional to the amount of DNA sample. Usually absorbance
is measured at 260 nm, at which wave length an absorbance of 1.0 corresponds to 50 µg of
double-stranded DNA per ml. UV absorbance can also be used to check the purity of a DNA
preparation. With a pure sample of DNA the ratio of the absorbencies at 260 nm and 280 nm
(A260/A280) is 1.8. Ratios of less than 1.8 indicate that the preparation is contaminated, either
with protein or with phenol.
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Special Types of DNA Extractions: Specific techniques must be chosen for isolation of
DNA from some samples. Typical samples with complicated DNA isolation are:
Archaeological samples containing partially degraded DNA.
Samples containing inhibitors of subsequent analysis procedures, most notably inhibitors
of PCR, such as humic acid from soil, indigo and other fabric dyes or haemoglobin in blood
Samples from microorganisms with thick cellular wall, for example yeast
Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily
isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in
insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble
fraction.
A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell.
The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only
low molecular weight mitochondrial DNA and any viral episomes present in the cell.
Instrumentation used in DNA Extraction A bead beater is used in the breaking apart or "lysing" of cells in the early steps of extraction
in order to make the DNA accessible.
Glass beads are added to an eppendorph tube containing a sample of interest and the bead
beater vigorously vibrates the solution causing the glass beads to physically break apart the
cells.
Other methods used for lysing cells include a french press and a sonication device.
A centrifuge such as this can spin at up to 15,000 rpm to facilitate separation of the different
phases of the extraction.
It is also used to precipitate the DNA after the salts are washed away with ethanol and or
isopropanol.
A gel box is used to separate DNA in an agarose gel with an electrical charge.
When the red and black leads are plugged into a power supply the DNA migrates through the
gel toward the positive charge due to the net negative charge of the molecule.
Different sized pieces of DNA move at different rates, with the larger pieces moving more
slowly through the porus medium, thereby creating a size separation that can be differentiated
in a gel.
4
Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
ADVANCED TECHNIQUES IN FIELD STUDIES: GENERAL CONCEPTS AND APPLICATIONS
PCR (POLYMERASE CHAIN REACTION)
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INTRODUCTION
Polymerase chain reaction, PCR, is an efficient and cost-effective way to copy or amplify
small segments of DNA without using a living organism.
It enables researchers to produce millions of copies of a specific DNA sequence in just a few
hours, yielding enough DNA required for analysis.
PCR can be used to amplify DNA samples as small as a single gene.
This innovative yet simple method allows clinicians to work using a minimal amount
of sample, such as blood or tissue.
This automated process bypasses the need to use bacteria for amplifying DNA.
PCR is a revolutionary method developed by Kary Mullis in the 1980s for which he received
the Nobel Prize in 1993.
PCR process can copy only short DNA fragments usually up to 10kb (1000bp).
The PCR reaction is carried out in a thermal cycler.
This is a machine that heats and cools the reaction tubes within it to the precise temperature
required for each step of the reaction.
Though PCR occurs in vitro, or outside of the body in a laboratory, it is based on the natural
process of DNA replication.
In its simplest form, the reaction occurs when a DNA sample and a DNA polymerase,
nucleotides, primers and other reagents are added to a sample tube.
The reagents facilitate the reaction needed to copy the DNA template.
There are three clear steps in each PCR cycle, and each cycle approximately doubles the
amount of target DNA.
This is an exponential reaction so more than one billion copies of the original or target DNA
are generated in 30 to 40 PCR cycles.
COMPONENTS OF PCR
DNA TEMPLATE - DNA template is the sample DNA that contains the target sequence –
DNA template is the region of DNA fragment to be amplified.
DNA POLYMERASE – “a type of enzyme that copies the region of DNA to be amplified”
or “a type of enzyme that synthesizes new strands of DNA complementary to the target
sequence”. The first and most commonly used of these enzymes is Taq DNA polymerase
(from Thermis aquaticus which grows in hot springs at a temperature of 90oC so Taq DNA
polymerase is not denatured at high temperature), whereas Pfu DNA polymerase
(from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA.
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Although these enzymes are subtly different, they both have two capabilities that make them
suitable for PCR: 1) they can generate new strands of DNA using a DNA template and
primers, and 2) they are heat resistant.
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TWO PRIMERS – Primers are short pieces of single-stranded DNA that are complementary
to the target sequence. Primers are short, artificial DNA strands (usually 18-25 bp)
nucleotides. They anneal (adhere) to the DNA template at the starting points where the DNA
polymerase can bind and begin synthesis. The polymerase begins synthesizing new DNA
from the end of the primer.
The melting temperature (Tm) of a primer should not be confused with the melting
temperature of the DNA. The melting temperature (Tm) of a primer increases with the length
of the primer. Primers that are too short would anneal at several positions along the length of
the long DNA template, which would result in non-specific copies. Melting temperatures that
are too high i.e. above 80oC can cause problems since the DNA-polymerase is less active at
such temperature. The optimal length of the primer is generally 20 – 40 nucleotides with a
melting temperature between 60 oC to 75 oC.
NUCLEOTIDES (dNTPs or deoxynucleotide triphosphates) - single units of the bases A,
T, G, and C, which are essentially "building blocks" for new DNA strands.
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SAMPLE PREPARATION
Before starting PCR, DNA must be isolated from a sample.
DNA extraction is a multi-step process that may be done manually or with an instrument like
the COBAS® AmpliPrep Instrument, the first instrument that prepared samples
automatically without human intervention.
Following sample preparation, the three-step PCR process is initiated.
PCR PROCESS OR PRINCIPLE OF THE PCR
The purpose of PCR is to make a huge number of copies of a DNA segment (gene). There are
3 major steps in a PCR, which are repeated. The PCR process consists of a series of 20 – 30
cycles. This is done in an automated cylinder which can heat and cool the tubes with the
reaction mixture in a very short time. The automated cylinder is also called thermal cylinder.
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DENATURATION - Separating the Target DNA – During the first step of PCR, called
denaturation.
The tube containing the sample DNA is heated to 94-96oC which separates the doublestranded DNA into two separate strands.
The high temperature breaks the relatively weak hydrogen bonds between the two DNA
strands.
This step is called denaturing.
Time given in this step is 1 – 2 minutes.
ANNEALING - Binding Primers to the DNA Sequence – After separating the DNA
strands, the temperature is lowered so the primers can attach themselves to the single
stranded DNA.
This step is called annealing.
During step two, the tube is cooled and primer binding occurs between 40oC- 60oC (104 –
140 degrees Fahrenheit).
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The temperature of this stage depends on the primers and is usually 5oC below their melting
temperature.
A wrong temperature may result in the primer not binding to the template DNA at all or
binding at random site.
PCR does not copy the all of the DNA in the sample.
It copies only a very specific sequence of genetic code, targeted by the PCR primers.
Primers are man-made oligonucleotides (short pieces of synthetic DNA) that bind, or anneal,
only to sequences on either side of the target DNA region.
Two primers are used in step two - one for each of the newly separated single DNA strands.
The primers bind to the beginning of the sequence that will be copied, marking off the
sequence for step three.
Step two yields two separate strands of DNA, with sequences marked off by primers.
The two strands are ready to be copied.
Time given in this step is 1 – 2 minutes.
EXTENSION - Making a Copy –
In the third phase of the reaction is called extension or elongation.
The temperature in the third step is increased to approximately 72oC (161.5 degrees
Fahrenheit).
The elongation temperature depends on the DNA polymerase.
Beginning at the regions marked by the primers, nucleotides in the solution are added to the
annealed primers by the DNA polymerase to create a new strand of DNA complementary to
each of the single template strands.
After completing the extension, two identical copies of the original DNA have been made.
The time for this step depends both on the DNA polymerase itself and the length of the DNA
fragment to be amplified. 1 minute per 1000bp.
PCR CYCLES
After making two copies of the DNA through PCR, the cycle begins again, this time using the
new duplicated DNA.
Each duplicate creates two new copies and after approximately 30 or 40 PCR cycles, more
than one billion copies of the original DNA segment have been made.
Because the PCR process is automated, it can be completed in just a few hours.
PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand.
Because DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, it
needs a primer to which it can add the first nucleotide.
This requirement makes it possible to delineate a specific region of template sequence that
the researcher wants to amplify.
At the end of the PCR reaction, the specific sequence will be accumulated in billions of
copies (amplicons).
At the beginning of the reaction, high temperature is applied to the original double-stranded
DNA molecule to separate the strands from each other.
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Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
REVERSE TRANSCRIPTION PCR (RT-PCR)
PCR is an efficient and cost-effective way to copy or amplify small segments of DNA. PCR
is commonly used to - Identify species, Identify alleles/genotypes to assess variability in a
population, create sequences for phylogenies to determine taxonomic relationships and also
to conduct forensic investigations.
Many viruses and other biological components (for example, mitochondria) utilize RNA as
their genetic material. RT-PCR differs from conventional PCR by first taking RNA and
converting the RNA strand into a DNA strand. Thus RT-PCR is a PCR test that is
designed to detect and measure RNA.
RT-PCR - (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA
into cDNA with enzyme reverse transcriptase. Thus RT-PCR stands for Reverse
Transcription-Polymerase Chain Reaction.
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RT-PCR is used to locate and quantify known sequences of mRNA in a sample.
The first step in RT-PCR uses reverse transcriptase and a primer to anneal and extend a
desired mRNA sequence.
If the mRNA is present, the reverse transcriptase and primer will anneal to the mRNA
sequence and transcribe a complimentary strand of DNA.
Thus the reverse transcriptase allows a single strand of RNA to be translated into a
complementary strand of DNA.
Once that reaction occurs, the routine PCR method is used to amplify the DNA.
That is the DNA strand is then replicated with primers and Taq Polymerase, and the standard
PCR protocol is followed.
This protocol copies the single stranded DNA millions of times in a small amount of time to
produce a significant amount of DNA.
The PCR products (the DNA strands) are then separated with agarose gel electrophoresis.
If a band shows up for the desired molecular weight, then the mRNA was in fact present in
the sample, and the associated gene was being expressed.
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The results from RT-PCR are used in two main ways.
First, RT-PCR shows whether or not a specific gene is being expressed in a sample.
If a gene is expressed, its mRNA product will be produced, and an associated band will
appear in the final agarose gel with the correct molecular weight for the gene.
Second, RT-PCR can quantify exactly how active the gene is in the sample.
This approach is used to identify how much mRNA is being produced by the gene.
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Advantage and Limitations of PCR and RT-PCR
The PCR reaction starts to generate copies of the target sequence exponentially.
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Only during the exponential phase of the PCR reaction is it possible to extrapolate back to
determine the starting quantity of the target sequence contained in the sample.
Because of inhibitors of the polymerase reaction found in the sample, reagent limitation,
accumulation of pyrophosphate molecules and self-annealing of the accumulating product,
the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a
"plateau effect" occurs.
This makes the end point quantification of PCR products unreliable.
PCR can amplify only DNA not RNA.
RT-PCR has been used to detect and study many RNA viruses.
It is a technique that allows the detection and quantification of mRNA. In order to meet
consumer and regulatory demands, several PCR-based methods have been developed and
commercialized to detect and quantify mRNA in various organisms.
It is a very sensitive method that shows whether or not a specific gene is being expressed in a
given sample.
RT-PCR is a very important test in the field of Genetically-Modified Organisms (GMO's)
because it gives researchers a mechanism to test whether any specific gene is turned on
(active) or turned off (inactive).
In nearly all published works, Next Generation Sequencing analyses of biodiversity usually
involve DNA extraction of bulk samples (mixtures of co-occurring taxonomic groups), PCR
amplification of targeted genetic markers, and NGS analysis for taxonomic composition.
PCR amplification of targeted genes is employed as the sole approach to acquiring sufficient
barcode sequences that are used for species identification.
In PCR-based metabarcoding approaches, various primer sets are used to amplify target DNA
fragments.
Primers designed to amplify the full range of taxa presented in the bulk sample are rarely
universal, it is thus difficult to predict the performance of primers when the investigated
fauna is largely unknown.
This almost always introduces taxonomic biases, which means that some organisms are easily
detected while others are constantly missed or under-represented.
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This artificial bias poses a serious problem for all biodiversity studies in which species
composition is important.
Also Amplification errors propagated during PCR, is potentially one of the major causes of
what is commonly known as “biodiversity inflation” or “false positives” found in nearly all
published NGS analyses of biodiversity.
This is the attribute of PCR that makes Real-Time Quantitative RT-PCR so necessary.
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Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
REAL-TIME RT-PCR
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The invention of PCR by Kary Mullis in 1984 was considered as a revolution in science.
The technology to detect PCR products in real-time, i.e., during the reaction, has seen a
dramatic increase in use over the past 2 years.
Real-time PCR (RT PCR) is thus becoming a common tool for detecting and quantifying
expression profiles of selected genes and series of RT PCR machines have also been
developed for routine analysis.
The high sensitivity and specificity of RT PCR allow it to be the first choice of scientists
interested in detecting dynamics of gene expression in plant/microbe associations.
The RT PCR allows quantitative genotyping and detection of single nucleotide
polymorphisms and allelic discrimination as well as genetic variations when only a small
proportion of the sample is carrying the mutation.
The use of multiplex PCR systems using combined probes and primes targeted to sequences
specific to counter partners of plant/ microbe associations is becoming more important than
standard PCR.
The multiplex RT PCR is suitable for multiple gene identification based on the use of
fluorochomes and the analysis of melting curves of the amplified products.
Although RT PCR is a powerful technique for absolute comparison of all transcripts within
the investigated tissue, it has a few problems as it depends critically on the correct use of
calibration and reference materials.
WORKING
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RT-PCR is an assay that monitors the accumulation of a DNA product from a PCR reaction
in real time.
This enables the researcher to quantify the amount of DNA in the sample at the start of a
reaction.
RT-PCR works on the same principle as the regular PCR.
To track the amplification of DNA in real times, probes are added to the reaction.
A probe is an oligonucleotide that is labelled with a florescent reporter and a quencher.
The single stranded DNA probe is designed to hybridise to a part of the DNA sequence.
In the single stranded DNA probe one of the nucleotide is labelled with a florescent tag
(florescent tag is conjugated to the terminal base).
Another nucleotide is labelled with a florescent quenching molecule (quencher is tagged to
the terminal base at the opposite end of the probe).
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The quencher decreases the fluorescence intensity as it rapidly absorbs the light energy
emitted by the florescence molecule as long as it remains in close proximity.
During the PCR cycle as the primers bind to the DNA strands the probe also finds the
complimentary strand between them.
The Taq polymerase joins nucleotides to the primer and starts polymerization. Polymerase
enzyme has both polymerase activity and nuclease activity. Thus when it encounters the
double stranded DNA in its path it will dissemble the strand that is in its way and release all
of the nucleotides of the probe and adds new nucleotides due to its polymerase activity.
As the nucleotides from the probe are released the fluorescent reporter and the quencher are
separated from one another.
In absence of the quencher molecule the fluorescent reporter can now emit fluorescent light.
Every time a amplicon is released another florescent marker is released therefore as the
number of amplicons doubles with each PCR cycle the amount of emitted florescent energy
also doubles.
This light generation is monitored by the PCR equipped with a flourometer.
With only one copy of the target DNA it takes 40 PCR cycles to cross the threshold of
detection and for the flourometer to detect the fluorescence. If the sample contains 32 times
more copies of the amplicons then the fluorescence can be detected 5 cycles earlier similarly
if the sample contains 1024 times more copies of the amplicons then the fluorescence can be
detected 10 cycles earlier
During the PCR cycle the probe denatures and anneals to the target sequence
For every amplification, of the target sequence, a florescent reporter is released from the
probe.
The RT-PCR has two main components – the Thermal Cycler (PCR machine) and an Optical
Module (to detect fluorescence in the tubes during the run).
The optical module measures the florescence intensity in the tube during the run.
The three steps in RT-PCR is Denaturation, Annealing and Elongation.
The reporter is only released when a DNA strand is completely polymerized by Taq
polymerase.
Cycle after cycle the DNA amplicons as well as the flouroscence intensity increases.
PCR Inhibitory Compounds
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The sampling procedures are of great importance towards the validation of analytical
methods for analysis.
The largest single source of error in the analysis of plant/microbe associations is the sampling
procedure.
Sampling risks can be managed by choosing an appropriate sample size for analysis.
The extraction and purification of nucleic acids is a crucial step for the preparation of
samples for PCR.
Current methods for gene expression studies typically begin with a template preparation step
in which nucleic acids are freed of bound proteins and are then purified.
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Many protocols for nucleic acid purification, reverse transcription of RNA and/or
amplification of DNA require repeated transfers from tube to tube and other manipulations
during which materials may be lost.
The data output from certain RT PCR machines gives an immediate appreciation of the
kinetics of the PCR occurring within the tube and, in addition, gives an instantaneous visual
representation of the amount of PCR product present following each cycle.
Sampling procedures are of great importance towards the validation of analytical
methods for analysis.
Protocols reported for the extraction of DNA/RNA from plant material, are complicated and
time consuming in application. The protocols should be perused case by case and to be
adopted judiciously for a particular plant species.
In this respect major variations exist in this step as compared to samples of mammalian (w.r.t.
animals) origin.
Isolation of RNA is particularly challenging because this molecule is sensitive to elevated
temperatures and is degraded by RNAses.
There are numerous commercially available kits for PCR. Detailed information is available
from the respective companies’ web-sites about the protocols and output information
generated.
SPECIES IDENTIFICATION
In plants, the presence of such a large number of multiple copies within each gene family
complicates the clear understanding of function of each member.
Plant molecular biologists prefer RT PCR methods to other methods.
Expression analysis of all members (33 genes) encoding cell-wall enzymes in Arabidopsis
thaliana using RT PCR revealed that most members exhibited distinct expression profiles
along with redundant expression patterns of some genes.
Similarly, an expression profile for shaggy-like kinase multigene family during plant
development has also been made using this technique.
A study using duplex RT PCR has also been described for determining the transgene copy
number in transformed plants with high degree of correlation with southern blot analysis.
Likewise, many studies are available on detection of copy number using RT PCR in various
crops.
ENVIRONMENTAL ISSUES
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RT PCR is a convenient method for detection of the mobility of genetic elements.
The worldwide increasing environmental pollution is pressing us to find new methods for
elimination of undesirable chemicals.
The application of microorganisms for the biodegradation of synthetic compounds
(xenobiotic) is an attractive and simple method.
Unfortunately, the majority of these pollutants are chemically stable and resistant to
microbial attack.
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The isolation of new strains or the adaptation of existing ones to the decomposition of
xenobiotic will probably increase the efficacy of microbiological degradation of pollutants in
the near future.
The cloning and expression in Escherichia coli of an ‘azo-reductase’ from various species
have been reported.
PLANTS
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RT PCR has been employed to study the gene expression patterns during several stresses
leading to activation of genes relating to signal transduction, biosynthesis, and metabolism.
Nitrogen deprivation response in Arabidopsis was analysed by profiling transcription factors
using Affymetrix ATH1 arrays and a RT RT-PCR platform.
The results revealed large number of differentially expressed putative regulator genes.
In this study, MapMan visualization software was used to identify coordinated, system-wide
changes in metabolism and other cellular processes.
Similarly, Czechowski and co-workers have profiled of over 1,400 Arabidopsis transcription
factors, and revealed 36 root and 52 shoot specific genes.
Further, gene expression studies have been made in the direction of stress signalling during
biotic and abiotic stress conditions in plants.
Standardization of house-keeping genes for such studies has been made in potato.
Among the seven common genes tested, ef1alpha was the most stable gene during biotic and
abiotic stress.
Data obtained by microarray analysis are questioned on few instances and confirmation is
achieved by RT PCR.
In general, studies made so far reveal a good relationship between these two techniques, and
for this reason RT PCR is considered as confirmatory tool for microarray results.
PLANT PATHOLOGY
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Host plant and associated microbes form a special consortium where the parasite is an alien
element.
Early diagnosis of the pathogens can provide rapid and suitable measurements for limiting
the epidemics and selection of appropriate control measures.
Molecular diagnostics is a rapidly growing area in plant pathology especially for detection
and quantification of commercially important crop pathogens.
As a novel methodology, adoption of RT PCR technique is of growing interest due to its
rapidity and sensitivity as well as its ability to detect minute amounts of the pathogen’s DNA
from infected plant tissues and insect vectors.
Simultaneous detection of several pathogens can be achieved by multiplex PCR. The
technique has aided detection of pathogens associated with serious diseases
like Fusarium head blight, which is a prerequisite for reduction in the incidence by
understanding of its epidemiology.
15
Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
DNA SEQUENCING
DNA sequencing is the determination of the precise sequence of nucleotides in a sample of
DNA. DNA sequencing enables us to perform a thorough analysis of DNA because it
provides us with the most basic information of all: the sequence of nucleotides.
POLYNUCLEOTIDE FORMATION
The 5' group of a nucleotide
triphosphate is held close to the
free 3' hydroxyl group of a
nucleotide chain.
The 3' hydroxyl group forms a
bond to the phosphorus atom of
the free nucleotide closest to the
5' oxygen atom. Meanwhile, the
bond between the first phosphorus
atom and the oxygen atom linking
it to the next phosphate group
breaks.
16
A new phosphodiester bond joins
the
two
nucleotides.
A pyrophosphate
group is
liberated.
The pyrophosphate group is
hydrolyzed (split by the addition
of water), releasing a great deal of
energy and driving the reaction
forward to completion.
Polynucleotides have a free 5'
phosphate group at one end and a
free 3' hydroxyl group at the other
end.
17
SANGER-COULSON’S “CHAIN TERMINATION METHOD”
Sequencing is the process by which you determine the exact order of the nucleotides in a
given region of DNA. With this knowledge, for example, we can locate the regulatory and
gene sequences and make comparisons between homologous genes across species and
identify mutations. In 1974, two methods were independently developed almost
simultaneously by an American team, led by A. Maxam and W. Gilbert, called “chemical
cleavage protocol”, while the English team, led by F. Sanger and A.R. Coulson designed a
procedure called “chain termination method”. Even though both teams shared the 1980
Nobel Prize, Sanger’s method became the standard because of its practicality.
THE SANGER-COULSON PRINCIPLE
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Sanger’s method, which is also referred to as dideoxy sequencing or chain termination
method, is based on the use of dideoxynucleotides (ddNTP’s) in addition to the normal
nucleotides (NTP’s) found in DNA.
Dideoxynucleotides are essentially the same as nucleotides except that they contain a
hydrogen group on the 3’ carbon instead of a hydroxyl group (OH).
These modified nucleotides, when integrated into a sequence, prevent the addition of further
nucleotides.
This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and
the next incoming nucleotide, and thus the DNA chain is terminated.
THE SANGER-COULSON METHOD
The classical chain-termination method requires a single-stranded DNA template, a
DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and
modified di-deoxynucleotidetriphosphates (ddNTPs. Before the DNA can be sequenced,
it has to be denatured into single strands using heat.
(a) Primer
 A short oligonucleotide primer is annealed to one strand of the template strands.
 This primer is specifically constructed so that its 3' end is located next to the DNA sequence
of interest.
18
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This primer will act as a starting point for complimentary strand synthesis reaction carried out
by the DNA polymerase.
Either this primer or one of the nucleotides should be radioactively or fluorescently labeled so
that the final product can be detected on a gel.
(b) Synthesis of the complementary strand
The chain synthesis is started by adding the enzyme plus each of the four deoxynucleotides
(dATP, dCTP, dGTP, dTTP).
In addition a single modified nucleotide is also included in the reaction mixture.
This is a dideoxynucleotide (e.g. dideoxy dCTP) which can be incorporated into the growing
polynucleotide strand just as efficiently as the normal nucleotide, but which blocks further
strand synthesis.
This is because the dideoxy-nucleotide lacks the hydroxyl group at the 3’ position of the
sugar component.
This group is needed for the next nucleotide to be attached; chain termination therefore
occurs whenever a dideoxynucleotide is incorporated by the enzyme.
(c) Four separate reactions result in four families of terminated strands
Once the primer is attached to the DNA, the solution is divided into four tubes labeled "G",
"A", "T" and "C".
Then reagents are added to these samples as follows:
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"G" tube: all four dNTP's, ddGTP and DNA polymerase
"A" tube: all four dNTP's, ddATP and DNA polymerase
"T" tube: all four dNTP's, ddTTP and DNA polymerase
"C" tube: all four dNTP's, ddCTP and DNA polymerase
As shown above, all of the tubes contain a different ddNTP present, and each at about onehundredth the concentration of the normal precursors (Russell, 2002).
As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA
polymerase.
However, on occasion a dideoxynucleotide is incorporated into the chain in place of a normal
nucleotide, which results in a chain-terminating event.
For example if we looked at only the "G" tube, we might find a mixture of the following
products:
Figure
1: An
example of the
potential fragments
that
could
be
produced in the
"G"
tube.
The
fragments are all
different
lengths
due to the random
integration of the
ddGTP's
19
 The strand synthesis reaction is carried out four times in parallel (ddATP, ddCTP, ddGTP and
ddTTP).
 The result will be four different distinct families of newly synthesized polynucleotide, one
family containing strands all ending in dideoxy ATP, one of strands ending in dideoxy TTP,
etc.
 The key to this method is that all the reactions start from the same nucleotide and end with a
specific base.
 Thus in a solution where the same chain of DNA is being synthesized over and over again,
the new chain will terminate at all positions where the nucleotide has the potential to be
added because of the integration of the dideoxynucleotides (Russell, 2002).
 In this way, bands of all different lengths are produced.
 Once these reactions are completed, the DNA is once again denatured in preparation for
electrophoresis.

The contents of each of the four tubes are run in separate lanes on a polyacrylamide gel in
order to separate the different sized bands from one another.
20
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After the contents have been run across the gel, the gel is then exposed to either UV light or
X-Ray, depending on the method used for labeling the DNA.
Figure 2: This is a polyacrylmide
gel of the reactions in the "G"
tube (the same sequences seen in
figure 1). The longer fragments of
DNA traveled shorter distances
than the smaller fragments
because of their heavier molecular
weight.The blue section indicates
the primer, the black section
indicates the newly synthesized
strand and the red denotes a
ddGTP, which terminated the
chain.
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The next step is to separate the components of each family so the lengths of each strand can
be determined. This can be achieved by very thin polyacrylamide gel electrophoresis.
In addition the electrophoresis is carried out at a high voltage, so the gel heats up to 60 0C and
above, making sure the strands do not re-assoiciate in any way.
Each band in the gel will contain only a small amount of DNA, so autoradiography has to be
used to visualise the results.
The label is introduced into the new strands by including a radioactive deoxynucleotide (32Por 35S-dATP) in the reaction mixture for the strand synthesis step earlier in the experiment.
As shown in Figure 2, smaller fragments are produced when the ddNTP is added closer to the
primer because the chains are smaller and therefore migrate faster across the gel.
If all of the reactions from the four tubes are combined on one gel, the actual DNA sequence
in the 5' to 3' direction can be determined by reading the banding pattern from the bottom of
the gel up.
Figure 3: This is an autoradiogram of a
dideoxy sequencing gel. The letters over
the lanes indicate which dideoxy
nucleotide was used in the sample being
represented by that lane. When you read
from the bottom up, you are reading
thecomplementary sequence
of
the
template strand.
It is important to remember though that this sequence is complementary to the template
strand from the beginning.
21
Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
DNA FINGERPRINTING
DNA fingerprinting is the method of identification based on an individual’s DNA. The
technique was developed by British geneticist Alec Jeffrey of Leicester University in 1984.
He noticed that certain sequences of DNA known as mini-satellites, do not contribute to the
functions of genes and are repeated within the genes. Jeffrey recognized that each individual
has a unique pattern of mini-satellites except in identical twins. These mini-satellites were
used as genetic markers to identify the individual.
TRADITIONAL DNA FINGERPRINTING
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In traditional DNA fingerprinting the DNA are collected from cells such as a blood sample
and cut into small pieces by using a restriction enzyme.
This generates thousands of DNA fragments of varying sizes.
The fragments are then separated on the basis of their size using gel electrophoresis.
The separated fragments are then transferred to a nitrocellulose or a nylon filter.
This is called a gel blot.
The DNA fragments within the blot are permanently fixed to the filter and the DNA is
denatured.
Radiolabeled probe molecules are then added that are complementary to the sequences in the
genome that contain repeat sequences.
These repeat sequences tend to vary in their length among variable individuals.
They are called variable number of tandem repeat sequences or VNTRs.
The probe molecule hybridizes to the DNA fragments containing the repeat sequence and
excess probe molecules are washed away.
The blot is then exposed to an x ray film.
Fragments of DNA that have hybridized with the probe appear as dark band on the x ray film.
Every sample show a different set of bands indicating that they came from different
individuals.
It is a test to identify and evaluate the genetic information - called DNA in a cell.
DNA fingerprinting is also called DNA typing, DNA profiling, genetic fingerprinting,
genotyping or identity testing.
It is the method of isolating and identifying variable elements within the base-pair sequence
of DNA.
DNA FINGERPRINTING (prevalent method)

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The most prevalent method of DNA fingerprinting used today is based on the polymerase
chain reaction and analyses variation at short tandem repeat regions of DNA, also known as
microsatellites.
These highly polymorphic regions have short repeated sequences of DNA.
22
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Because different people have different numbers of repeat units, these regions of DNA can be
used to differentiate individuals.
These repeat locations are targeted with sequence-specific primers and are amplified.
The DNA fragments that result are then separated and detected using electrophoresis; instead
of using radioactive probes, the gel is scanned directly and the DNA profile uploaded directly
into a computer.
PROCEDURE
The procedure for creating a DNA fingerprint consists of first obtaining a sample of cells
which contain DNA.
In Jeffreys’s original approach, which is based on restriction fragment
length polymorphism (RFLP) technology, the DNA is extracted from the cells and purified.
The DNA is then cut at specific points along the strand using special restriction enzymes.

RFLP focuses on segments that contain sequences of repeated DNA bases, which vary widely
from person to person.
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The segments are separated using a laboratory technique called electrophoresis, which sorts
the fragments by length.
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The segments are radioactively tagged to produce a visual pattern known as an
autoradiograph, or "DNA fingerprint," on X-ray film.
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A newer method known as short tandem repeats (STR) analyses DNA segments for the
number of repeats at 13 specific DNA sites.
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The chance of misidentification in this procedure is one in several billion.
The assay developed by Jeffreys has been supplanted by approaches that are based on the use
of the polymerase chain reaction (PCR) and so-called microsatellites (or short tandem
repeats, STRs), which have shorter repeat units (typically 2 to 4 base pairs in length) than
minisatellites (10 to more than 100 base pairs in length).
Thus in yet another process, PCR is used to produce multiple copies of segments from a very
limited amount of DNA, enabling a DNA fingerprint to be made.
PCR amplifies the desired fragment of DNA (e.g., a specific STR) many times over, creating
thousands of copies of the fragment.
It is an automated procedure that requires only small amounts of DNA as starting material
and works even with partially degraded DNA.
Once an adequate amount of DNA has been produced with PCR, the exact sequence of
nucleotide pairs in a segment of DNA can be determined by using one of several biomolecular sequencing methods.
The enzymes produced fragments of varying lengths that were sorted by placing them on a
gel and then subjecting the gel to an electric current (electrophoresis): the shorter the
fragment, the more quickly it moved toward the positive pole (anode).
The sorted double-stranded DNA fragments were then subjected to a blotting technique in
which they were split into single strands and transferred to a nylon sheet.
The fragments underwent autoradiography in which they were exposed to DNA probes—
pieces of synthetic DNA that were made radioactive and that bound to the mini-satellites.
A piece of X-ray film was then exposed to the fragments, and a dark mark was produced at
any point where a radioactive probe had become attached.
The resultant pattern of marks could then be analysed.
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23
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Automated equipment has greatly increased the speed of DNA sequencing and has made
available many new practical applications, including pinpointing segments of genes of
interest.
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Once a sufficient sample has been produced, the pattern of the alleles from a limited number
of genes is compared with the pattern from the reference sample.
APPLICATIONS
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The DNA profile of every individual is unique. It can never be identical even in biologically
related individuals except in identical (monozygotic) twins. The technique of DNA
fingerprinting has revolutionized the identification of individual organisms and species. The
use of these techniques does not alter the DNA of the organism and does not involve genetic
engineering.
An early use of DNA fingerprinting was in legal disputes, notably to help solve crimes and to
determine paternity.
The technique was challenged, however, over concerns about sample contamination, faulty
preparation procedures, and erroneous interpretation of the results. In addition, RFLP
required large amounts of high-quality DNA, which limited its application in forensics.
Some of the concerns with DNA fingerprinting, and specifically the use of RFLP, subsided
with the development of PCR- and STR-based approaches.
DNA fingerprinting technology has made it possible to identify the source of biological
samples found at scenes of crime.
This will resolve disputes of forensic wildlife, protection of farmer’s rights and biodiversity.
This remarkable technology provides exclusion as well as positive identification with
virtually 100% precision.
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The technique can also be used to establish paternity.
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The techniques used in DNA fingerprinting also have applications in palaeontology,
archaeology, various fields of biology, and medical diagnostics.
It has, for example, been used to match the goat skin fragments of the Dead Sea Scrolls. In
biological classification, it can help to show evolutionary change and relationships on the
molecular level, and it has the advantage of being able to be used even when only very small
samples, such as tiny pieces of preserved tissue from extinct animals, are available.
DNA fingerprints have been used to identify individuals in criminal cases, cases of disputed
parentage and victims or warfare or accidents. DNA fingerprints are also used for identifying
pathogens including viruses, bacteria and parasites. Individual plants, animals, fungus or alga
and their progeny may be traced using DNA fingerprints.
DNA fingerprint is designed to identify individuals or clones. The method soon spread to the
study of genetic lineages of wild and domestic animals and plants.
There are numerous examples of the use of DNA fingerprinting in agriculture.
DNA fingerprinting traced the origin of a patented yellow bean variety Enola to an older
Mexican variety, and Enola clearly lacked differences to distinguish it from the traditional
Mexican cultivar.
DNA fingerprinting identified the fruit tree species used in selection and introgression of new
varieties.
DNA fingerprinting of microbial plant pathogens proved useful in diagnosis and disease
management.
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24
Dr. M. SAHA
Associate Professor
Botany Department
M.Sc. I (BIODIVERSITY)
Sem II
PSBWCM204
UNIT I
SOUTHERN BLOTTING AND ITS APPLICATIONS
The technique was developed by E. M. Southern in 1975. The Southern blot is used to verify
the presence or absence of a particular DNA fragment in a sample. In this procedure the DNA
is isolated and then digested with a specific restriction endonuclease enzyme. Then the DNA
restriction fragments are loaded on the Agarose gel and the fragments are separated by
electrophoresis according to size. The smaller fragments migrate faster across the gel than the
larger fragments. The DNA is then transferred from the fragile gel to a nylon membrane.
Next the radioactively labelled nucleic acid probes are added. The probes bind to the
complementary DNA segments. To detect the position of radioactive probe the nylon
membrane is covered with an X ray Film. After development the position of the probe
becomes visible as dark bands.
PRINCIPLE
The principle of this technique is hybridization. Hybridization is a procedure of finding out a
specific DNA molecule by using a complementary strand or a complementary DNA probe
that will bind with the target sequence and form a hybrid.
PROCEDURE:
1.
2.
3.
4.
5.
Extraction and Purification of the DNA from the cell
DNA is restricted with enzymes
Separated by electrophoresis
Denature DNA
Transfer to Nitrocellulose paper (Blotting)
25
6. Add labelled probe for hybridization to take place
7. Wash off un-bound probe
8. Autoradiograph
STEP 1: DNA ISOLATION AND PURIFICATION
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The first step is to isolate the DNA from cell.
The cells are places in a cell lysis buffer.
Usually the cells are incubated with detergent to promote cell lysis.
Lysis occurs as the cell content is released.
Lysis frees cellular proteins and DNA.
In the next step protein should be removed from the sample.
In this purification procedure proteinase enzymes are used as the proteins released
have to be degraded.
Thus proteins are enzymatically degraded by incubation with proteinase.
DNA is purified from solution by alcohol precipitation.
It can either be ethanol or methanol.
Visible DNA fibres are removed and suspended in a buffer solution.
STEP 2: RESTRICTION DIGESTION
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This DNA is cut into small fragments with the use of restriction endonuclease enzyme
(RE) like EcoR1
The restriction endonuclease enzyme cuts the DNA at a particular restriction size.
This generates DNA fragments of different size
STEP 3: GEL ELECTROPHORESIS
These fragments are separated by gel electrophoresis.
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Gel Electrophoresis is a separation technique.
It is the separation of DNA fragments is based on size of the. DNA fragments
Gels are solid Agarose or Poly acrylamide, with microscopic pores through which the
DNA can travel.
The smaller fragment migrate faster than the larger fragments of DNA in the agarose
gel.
The procedure is that an electric current draws the negatively charged DNA through
the gel towards the positively charged electrode.
Gel is soaked in a buffer.
Standards should also be run.
For visualization staining is done with Ethidium bromide.
Under UV florescence the bands can be seen.
STEP 4 AND 5: DENATURATION AND BLOTTING
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Since the DNA is double stranded molecule the DNA should be denatured.
26
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Denaturation of DNA is often carried out with an alkaline solution such as NaOH.
DNA is then neutralised with NaCl to prevent re-hybridization before adding the
probe.
Blotting is the transfer of the DNA bands from the gel to a nitrocellulose membrane.
This blot is made permanent by exposing to UV radiation or drying at 80oC
The DNA bands in the gel and the nitrocellulose sheet is placed above the gel. Later
we can see that these bands are transferred from the gel to the nitrocellulose
membrane and the process is called blotting.
STEP 6: HYBRIDIZATION
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Hybridization is done as we need to find out if the target DNA is present in the
sample or not.
The labelled probe is added to the membrane in buffer and incubated for several hours
to allow the probe molecule to find the target DNA fragments.
Probes are small pieces of labelled DNA used to find the complementary DNA
fragment.
That is why the principle is called hybridization.
The probe binds with the complementary DNA fragment, as the probe radiolabelled it
can be detected.
STEP 7: WASHING AND AUTORADIOGRAPHY
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Excess un-bound probes are washed out.
For radioactive probes, a X ray film is placed over the membrane
If the sample contains DNA of interest then the probe will emit radioactivity on the
XRay film the emission of radiation will make bands on the XRay film indicating that
our DNA of interest is present
After development there will be dark bands on the film where ever the probe bound
APPLICATIONS
1.
2.
3.
4.
5.
6.
7.
To identify specific DNA in a sample.
To isolate desired DNA for making the rDNA.
Used in the prognosis of cancer and prenatal diagnosis of genetic diseases.
In RFLP
Used in Phylogenetic analysis
Diagnosis of HIV-1 and infectious diseases
Applications of DNA fingerprinting include:
 Paternity testing
 Criminal identification and forensic Science
 Personal identification
(all diagrams are internet download)