ABSTRACT

Cancer is a deadly disease that causes millions of deaths every year all around the world.
Most people are diagnosed with cancer when it has reached its severe stage and its cure
becomes almost impossible and that costs them their lives. But with the advancements in
medical technology, we now have various techniques that can be used to detect, diagnose,
treat and even prevent cancer that have proved to be a boon for the cancer patients. There are
many techniques like PCR (Polymerase Chain Reaction), RT-PCR (abbreviated as Reverse
Transcription PCR), RFLP (abbreviated as Restriction Fragment Length Polymorphism), Gel
Electrophoresis (agarose, native and acrylamide gels), DNA extraction through various
sources like through blood sample, hair strand, tissue, scrape sample, cell lines, etc. Using
these techniques, various disputes can also be resolved like the legal cases of paternity. Also,
for detecting various genetic diseases, for studying gene sequences, etc. these can be used
extensively.

In this report there is a clear-cut description about how above-mentioned techniques are used
and the way they perform their functions.
INTRODUCTION
Malignant growth (Latin word – "Crab", which means Malignancy) alludes to a gathering of
illnesses brought about by uncontrolled and unusual development of cells that can happen in
any piece or part of the body. These cells will in general multiply and metastasize in an
uncontrolled way to shape a tumor (piece of tissues) or state a threatening development.
Malignant growth can include practically any tissue of the body and can have numerous
particular structures in every single territory of body.

The recurrence of a specific sort of disease may rely upon sexual orientation, for eg. – Skin
malignancy is regular in the two people (man and woman) yet prostate disease happens in
guys while bosom malignant growth happens significantly in females. Malignancy recurrence
does not liken to disease mortality since certain tumors like skin malignant growth are
effectively reparable while some like Lung disease are the main source of passings for the
two guys and females in USA in the present date.

Cancerous cells are very much or totally different from the normal cells of body and the
various distinctive properties of cancerous cells involve:

 Loss of contact inhibition: normal cells show a property called contact inhibition, in
which the dividing cells when in contact with other cells inhibit their uncontrolled
growth but cancer cells tend to have lost this property. Instead the cancerous cells
continue to divide, giving rise to a mass of cells called neoplasm or tumor.
 Cancer cells are immortal and have shorter population doubling time
 Variation in cell size and shape
 Small cytoplasmic volume relative to nucleus
 Cellular and nuclear polymorphism
 Exhibit abnormal mitosis and chromosomal abnormalities
 Malignant cells usually have high levels of hexokinase increasing their glucose
utilization
 Cancerous cells are Anchorage independent
 They are invasive and genetically not stable
 Large, variably shaped nuclei
 Changes in cell membrane and organelles, etc.
 On the histological basis, cancer can be divided into following major categories:

1. CARCINOMA (malignant neoplasm of epithelial origin. Eg- breast cancer, skin

cancer, oesophageal cancer, lung cnacer, vaginal cancer, etc.)
2. SARCOMA (cancer of supportive and connective tissues. Eg- Angiosarcoma,
osteosarcoma, chondrosarcoma, fibrosarcoma, glioma, etc.)
3. MYELOMA (cancer that originates in the plasma cells of bone marrow. Eg- 15% of
all blood cancers are myelomas)
4. LEUKEMIA (group of cancers grouped within blood cancers, often associated with
overproduction of white blood cells)
5. LYMPHOMA (develop in glands or nodes of lymphatic system. Eg- Hodgkin and
Non- Hodgkin Lymphoma)

HALLMARKS OF CANCER:

 Sustaining proliferative signaling

 Evading growth suppressors
 Activating invasion and metastasis
 Enabling replicative immortality
 Inducing angiogenesis
 Resisting cell death

There are various techniques which are used in the diagnosis, imaging and treatment of
cancer which are performed in pathological labs and also in research laboratories. The
following content will be based on those techniques only and how the procedures are carried
out, what all equipments are used and what reagents are required has all been mentioned in
the later pages.
REVIEW OF LITERATURE

Equipments used in laboratory:

1. CENTRIFUGE:
 An equipment used in the laboratory to carry out centrifugation.
Centrifugation is a technique which is used for isolating and analyzing cells,
subcellular fractions, supramolecular complexes and isolated macromolecules
such as proteins or nucleic acids.
 Principle: Application of the centrifugal force for the separation of different
particles in a solution on the basis of their size, shape, density, rotor speed and
viscosity of the medium.
 Use: Centrifugation technique is employed to separate two miscible
substances but also to analyze the hydrodynamic properties of the
macromolecules.
 How shape, density and size affect centrifugation – Shape dictates the rate of
sedimentation or centrifugation (globular particles tend to settle down faster
than other shaped particles), particles with higher density settle down faster
than those with lower density float, particles with more size are easily
centrifuged.
2. SPINNER:
 Sometimes when we add a reagent solution to a sample in very small amounts
like for example 4 microliters is added to a sample, then it may condense or
say remain on the wall of the tube containing the sample.
 So, a simple device or equipment called spinner is used in the laboratory to
ensure that any condensed droplets on the wall of the tube is returning to the
bottom of the tube where it has to mix with the sample or simply it should be
in contact with the sample.
3. VORTEX MIXER:
 It is a simple device or an equipment which is commonly used in the
laboratories to mix small vials of liquid at very high speed.
 It consists of an electric motor with the drive shaft oriented vertically and
attached to a (cupped) rubber piece which is mounted slightly off center.
4. UV TRANSILLUMINATOR:
 It is a device or an equipment which is used after electrophoresis is done.
 In order to view the samples of DNA, RNA, proteins and their precursors in
gel electrophoresis, a UV trans-illuminator can be used.
 It emits high levels of UV radiations through the viewing surface.
 The samples can then be visualized in the trans-illuminator in the dark
surroundings.
5. pH METER:
 As the name indicates, a pH meter is used to measure the pH of solutions
based on the H+ ions activity in water based solutions, indicating the acidity
or alkalinity expressed as pH.
 It consists of either two electrodes (Glass electrode and a Reference electrode)
or a combination electrode.
 A pH meter measures the voltage difference between the two electrodes and
then displays the result converted into corresponding pH value.

6. AUTOCLAVE:
 It is a device that works with a combination of steam (temperature), pressure
and time, and autoclave serves as a physical method for disinfection as well as
sterilization.
 They operate at high temperature and pressure in order to kill the
microorganisms and spores also.
 It generally works on the principle that Boiling point is inversely related to
pressure at constant volume. While pressure is directly proportional to
temperature.
 So, for about 15 psi pressure, temperature generally rises upto 121 degree
Celsius.
7. DRY BATH:
 This device is often used in molecular biology, biochemistry and genetic
applications. It is an equipment which is used to heat samples or a
 “microprocessor controlled” (digital) devicefor warming up the samples and
liquids upto preset or body temperature (which is 37 degree).
 It is also known as “dry block heater”.
 It has a vital role in incubation and activation of cultures, enzyme reactions,
coagulation study, inactivation of serum (sera), restriction digestion and
Polymerase Chain Reaction (PCR) etc.
8. INCUBATOR:
 It is a very important equipment which is used in all biological or
biotechnological laboratories.
 It is used to grow and maintain microbiological cultures or simply the cell
cultures.
 It provides optimum conditions like temperature, humidity, and other
conditions like CO2 as in case for the CO2 incubators in which the purpose of
the gas is to maintain the pH of the sample and the surroundings.
9. LAMINAR HOOD:
 A laminar flow hood is a safety chamber or a safety cabinet that ensures the
protection of the biological sample (i.e. the product), the personnel and the
environment.
 It prevents contamination of the products.
 These are generally equipped with the HEPA (High Efficiency Particulate Air)
filters which are responsible for the flow of HEPA- filtered air towards the
product and personnel for their safety.
 Even the air released into the environment is also HEPA- filtered.
 Based on the position of the HEPA filters, the laminar hood can be either
vertical or horizontal.
10.MICROPIPETTE:
 An extremely fine pipette for estimating, moving or infusing exceptionally
little amounts of fluids, for example, in microliters. These are most commonly
used in chemistry, biology, forensics, bio-technology, cancer, pharmaceutical,
and drug discovery labs among others.
 These are most commonly used in chemistry, biology, forensics, bio-
technology, cancer, pharmaceutical, and drug discovery labs among others.
  They come in different sizes for the different sizes of the tips (that can contain
different volumes) and the size may include along the volumes:
a. P2 (0.2-2 microliters)
b. P10 (1-10 microliters)
c. P20 (2-20 microliters)
d. P100 (20-100 microliters)
e. P200 (20-200 microliters)
f. P1000 (100-1000 microliters)

Use a disposable pipette tip every time to aspirate liquid, a tip is the only part of the pipette
that makes contact with the solution. A new tip is utilized for every sample to prevent cross
contamination.

11.EPPENDORF TUBES:
 These are the small transparent tubes in which the sample is placed usually
while working with the help of micropipettes.
 These are often called as the ‘centrifuge tubes’.
 They have labelling/markings (in ml) on the outside of their walls for taking
the proper volume of liquid/sample.
 As they come in various sizes, these tubes are also used for PCR.
12. WEIGHING BALANCE:
 It is used for measuring small mass in sub milligram or gram range.
 Samples are placed inside the weighing balance in a small piece of paper
(should be absolutely clean) or a small light weighted container and weight of
the latter is tared, so only the sample is weighed.
 Measuring pan of a weighing balance is inside a ‘transparent enclosure with
doors so that the dust does not collect and so any air currents in the room do
not affect the working of the weighing balance machine’.

13.GEL DOC:
 A Gel Doc is a device that can be considered as an alternate to the UV Trans-
illuminator.
  When the electrophoresis is done and the DNA sample has migrated towards
the opposite charge, then for the visualization of the DNA bands, Gel doc is
used.
 The ready to visualize sample is placed inside the Gel doc device which is
attached to a computer system.
 Then through computer, command is given and the sample is visualized and
subsequently the image may be captured if needed for further reference.
 Also the illumination can be adjusted.

14.HEATING OVEN:
 It can be used for different or various purposes like sample drying, baking,
annealing, conditioning, sterilizing, evaporating, mixing, dehydrating and for
other general laboratory works.
 The operating temperature range up to 235 degree Celsius for some while it
can be up to even 300 degree Celsius for some.
 REAGENTS USED:

Various reagents used along with their composition and functions:

S.No. REAGENT COMPOSITION FUNCTION

1. TAE buffer Tris base  Maintains the pH of
Glacial Acetic Acid the solution in the
EDTA (pH 8) range 7-9.
Distilled water  TAE should be used
for larger DNA
fragments.

2. TBE buffer Tris base  Maintains pH in the

Boric Acid (or Borate) range of 8-8.5.
EDTA (pH 8)  Borate is an enzyme
Distilled water inhibitor, resolves <
2kb fragments better.
 So it is used for
smaller fragments.
3. PBS (Phosphate NaCl  Helps in maintaining a
Buffered Saline) KCl constant ph.
Na2HPO4  The osmolarity and
KH2PO4 ion conc. of the
Distilled water solution usually match
with those of human
body (isotonic).
4. Lysis buffer (whole NH4Cl  It is used for the lysis
blood) KHCO3 of the RBCs and does
EDTA (pH 8) not affect WBCs from
Double Distilled water which DNA is
(autoclaved) extracted.
5. PAGE MQ  Used for analyzing
 TBE protein mixtures
Acrylamide qualitatively.
APS  Useful in monitoring
TEMED protein purification.
 Can also be used for
determining the
relative molecular
mass of proteins.
6. APS Ammonium persulfate  Oxidizing agent, often
Distilled water used along with
TEMED in order to
catalyze the
polymerization of
acrylamide and bis-
acrylamide to prepare
the polyacrylamide
gels for
electrophoresis.
7. Lysis buffer (Tissue Tris EDTA  For solubilizing the
biopsy/ scrapes) SDS cell membrane and
Autoclaved double breaking down the
distilled water cell wall.
 Also causes protein
denaturation.
8. EDTA (pH 8) EDTA  Acts as chelating
Distilled water agent.
NaOH  Binds with metal ions
like Mg2+ which are
required for the action
of DNases.
 So, they prevent the
activity of DNases.
9. Ammonium Chloride NH4Cl  Used for the gentle
 (NH4Cl) Distilled water lysis of erythrocytes,
with minimal effects
on leukocytes.
10. Sodium Acetate (pH Sodium acetate  It helps in the
5.2) Autoclaved double precipitation of DNA
distilled water that is visible as a
white ppt.
 The Na+ ions bind to
the phosphate group
of DNA.
11. PCR buffer MgCl2  This buffer is
KCl necessary for the
Tris HCl activity of heat stable
Distilled water enzyme Taq
polymerase (obtained
from Thermus
aquaticus)

Table 1: Reagents used in laboratory

DNA

DNA stands for deoxyribonucleic acid. It is a very important nucleic acid which is
found in all living organisms (except RNA viuses). Usually it is found in nucleus of
the cell and called genomic DNA. But it can also be present in mitochondria (both
plant and animal cell) and in plastids (in plant cell). DNA consists of long nucleotide
chains. A nucleotide is made up of three components:
(i) Deoxyribose sugar (in case of DNA)
(ii) Phosphate group
(iii) Nitrogenous bases

 The phosphate group provides overall negative charge to DNA.

 The nitrogenous bases are of two types- Purines and Pyrimidines.
  The purines consist of Adenine (A) and Guanine (G).
 The pyrimidines consist of Cytosine (C), Thymine (T), Uracil (U).
 DNA contains A, T, G, C while U is present in RNA (Ribonucleic Acid) in place of
T.

Fig. 1 - Structure of a nucleotide

TECHNIQUES

1. DNA EXTRACTION

DNA can be extracted through various methods and the protocol for each method is different.
One of the most widely used method for DNA extraction is Phenol- Chloroform method.

Why phenol and chloroform?

The whole concept is based on “like dissolves like”. As we know that polar molecules
dissolve in polar solvents whereas the non- polar molecules dissolve in non- polar solvents.

BASIC PRINCIPLE: We know that water is polar while phenol is non- polar. The density
of water is 1g/cm3, density of phenol is 1.07 g/cm3, and the density of chloroform is 1.49
g/cm3. So, phenol is denser than water. And as per our knowledge, DNA is polar (as it carries
a net negative charge on its backbone). This method separates DNA molecules on the basis of
their “solubility in immiscible solutions” and this is the basic principle of phenol- chloroform
DNA extraction method.

 The reagents used in the phenol- chloroform extraction of DNA are as follows:
(a) PHENOL: It is non-polar so it cannot and it will not mix with water. So, when
together these will remain as separated phases or layers with phenol being at
bottom because of its higher density than water. DNA is insoluble in phenol.
Protein has both polar and non-polar groups as it is a long chain of different
amino acids. Polar is hydrophilic while non-polar is hydrophobic. The bonds
between the amino acids are broken down by the addition of phenol. When the
sample containing DNA is mixed with phenol, it forms a foam like emulsion.
(b) CHLOROFORM: It allows the proper separation of organic and the aqueous
phase which are formed after centrifugation. It also denatures the lipids in the cell
suspension.
(c) ISOAMYL ALCOHOL: It is an anti- foaming agent. It helps in reducing foaming
at the interphase of the organic and aqueous phase. Usually it is used along with
chloroform in the process of DNA extraction.
(d) TE buffer: This buffer is used to dissolve DNA and also to maintain the pH of the
solution.
(e) SDS: SDS stands for Sodium Dodecyl Sulfate. It is an anionic detergent which is
used to denature the proteins. It also provides an overall negative charge to the
protein irrespective of the native charge present on the protein. It also disrupts the
cell membrane and destabilizes all the hydrophobic interactions holding the
macromolecules in their native form.
(f) PROTEINASE K: This is a broad spectrum serine protease that cleaves the
peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino
acids. It is stable over a wide pH range. So, basically the endopeptidase enzyme
proteinase K digests the contaminating proteins and protects the nucleic acid i.e.
DNA from the nuclease attack also.
(g) TISSUE/ CELL LYSIS BUFFER: As the name indicates, the lysis buffer is
required for the lysis of the cells so that the inside contents of the cell can come
out and desired material can be extracted. Generally,the lysis buffer contains SDS
for DNA extraction.
 (h) ISOPROPANOL: It has two main functions while DNA extraction and these are,
first is it removes water from the surroundings of DNA and the other function is
that it decreases the solubility of DNA so that it can easily be precipitated.
(i) SODIUM ACETATE: It has a role in the precipitation of DNA. Sodium binds
with the negative phosphate group and this causes precipitation of DNA.

DNA extraction through cell lines

Cell lines – SiHa and HeLa (both are cervical cancer cell lines).

PROTOCOL:

i. Take the cell line samples into two separate eppendorfs. Add 1X TE
buffer, cell lysis buffer, Proteinase k in appropriate amounts and
incubate overnight at 45° C in water bath (70 cycles/min).
ii. Next day, add equal volume of Tris- EDTA equilibrated phenol. Mix
by gently inverting each tube for 10-15 minutes. Then centrifuge
@10,000 rpm for 8 minutes.
iii. After centrifugation, two layers are formed. The above one is aqueous
layer while the lower denser layer is the organic layer (organic phase)
composed of phenol. DNA is present in the aqueous layer.
iv. Take the supernatant (aqueous layer) into new tubes using micropipette
and discard the tubes with organic phase.
v. Now add equal volumes of phenol and CIA (CIA is Chloroform and
Isoamyl alcohol) in the ratio 1:1 where CIA is in the ratio 24:1.
vi. Mix by inverting and slight shaking the tubes for 10-15 minutes or put
in the overhead shaker for 10 minutes. Centrifuge @10,000 rpm for 8
minutes.
vii. Again, collect only the supernatant in fresh eppendorfs and discard the
previous tubes.
viii. Add equal volume of CIA where CIA is in the ratio 24:1. Put in
overhead shaker for 10 minutes or do it manually for 10-15 minutes.
Centrifuge @ 10,000 rpm for 8 minutes.
ix. Again, collect the supernatant only in fresh tubes.
x. Add equal volume of chilled isopropanol and 1/10th volume of chilled
3M sodium acetate.
 xi. Mix it gently.
xii. White threads can be seen on mixing. These threads are DNA only.
xiii. Now place your tubes with white precipitated threads into refrigerator
at -20° C overnight.
xiv. Next day, centrifuge the samples @12,000 rpm for 45 minutes.
xv. Discard the supernatant without disturbing the pellet.
xvi. Add 200 µL of 70% ethanol (chilled) to each tube. Put on vortex.
Resuspend the pellet by tapping it. Then centrifuge @ 10,000 rpmfor 8
minutes.
xvii. Discard the supernatant again without disturbing the pellet. Again add
200 µL of 70% ethanol to each tube, put on vortex and resuspend the
pellet by tapping it.
xviii. Centrifuge @10,000 rpm for 8 minutes. Discard the supernatant.
xix. Put the tubes open on dry bath at 37°C and allow the ethanol to
evaporate completely.
xx. Once the ethanol is completely gone, add 10-20 microliters of TE
buffer to each tube depending on the size of the pellet.
xxi. Now put the close tubes again on the dry bath at 37° C overnight for
the proper mixing of DNA with the buffer.
xxii. Next day whenTE buffer is mixed properly with DNA, perform the
electrophoresis step. Use 1% agarose gel to visualize the DNA
extracted.

DNA extraction through blood

DNA can only be extracted from the WBCs and not from the RBCs as the latter ones lack
nucleus.

PROTOCOL:

i. Using a syringe, collect 3 ml blood (from vein) in a falcon tube already

containing some EDTA.
ii. Keep it still for 2-3 hours on stand. Then there will be observed two layers
which will be plasma and serum (upper and lower respectively) in the
tube.
 iii. Remove the upper layer of plasma in a separate Eppendorf using
micropipette.
iv. From the layer of serum, take 300 µL in another tube. To it, add 900 µL
(thrice its volume) blood lysis buffer or RBC lysis buffer. Mix by gentle
shaking. Centrifuge @10,000rpm for 5 min.
v. On centrifugation, there appears a precipitated white pellet. Discard the
supernatant (red serum).
vi. Again add 900 µL blood lysis buffer. Mix well and resuspend the pellet
by tapping it so that it properly dissolves in buffer.
vii. Centrifuge @10,000 rpm for 5 minutes. Discard the red supernatant.
viii. Repeat step vi and vii one more time.
ix. Then add 300 µL sodium- EDTA (pH 8) to the cell pellet. Vortex it for
proper mixing.
x. Add 30 µL (1/10th of original volume which was 300 µL) SDS. Mix well.
xi. Add 6µL of Proteinase K to the Eppendorf tube.
xii. Seal air tight using parafilm tape.
xiii. Incubate in water bath (45° C, 70 cycles/min) overnight.
xiv. Next day remove the tape and perform the Phenol- Chloroform method of
DNA extraction i.e. follow steps ii. to xxii. of previous DNA extraction
(from cell lines).

FIG. 2: DNA precipitated by sodium acetate

DNA extraction from Cervical Scrape

PROTOCOL:

i. Hold the brush inside cervical scrape container with the help of micropipette with a
tight tip and mix well the tissues in the scrape sample.
ii. Then transfer the equal volume of sample in two new Eppendorf tubes.
iii. Centrifuge @ 10,000 rpm for 6 minutes.
iv. Keep the pellet without disturbing and discard the supernatant.
v. WASHING- Add 500 µL PBS to each tube. Centrifuge @ 10,000 rpm for 6 minutes.
Discard the supernatant.
vi. Repeat step v one more time.
vii. After discarding the supernatant in second washing with PBS, add 400 µL of TE
buffer to each tube.
viii. Then add 200 µL of tissue lysis buffer to each tube.
ix. Add 18 µL of Proteinase K (diluted with distilled water) to each tube.
x. Cover tightly with parafilm tape. Put in water bath overnight at 45° C.
xi. Next day remove the tape and perform the Phenol- Chloroform method of DNA
extraction i.e. follow steps ii. to xxii. of previous DNA extraction (from cell lines).

2. GEL ELECTROPHORESIS:

Electrophoresis describes the migration of charged particles under the influence of an electric
field. Many important biomolecules like nucleic acids (DNA, RNA), proteins, etc. exist as
electrically charged species (either cations or anions) and depending on their net charge, these
will migrate to cathode or anode under the influence of applied electric field.

Two items are required for electrophoresis apparatus and these are:

1. An electrophoresis unit
2. A power pack

The electrophoresis units are available for either vertical or horizontal gel systems. It
means that generally the horizontal gel systems are used for DNA samples using agarose
gels while the vertical gel system is used for Protein sample using polyacrylamide gels.
The vertical gel system can also be used for RFLP.

HOW WE PERFORM ELECTROPHORESIS:

1. Set the casting tray and apply combs to it (one comb usually can make 8 wells for
sample loading).
2. Prepare gel- suppose we are preparing 1% agarose gel, take 0.5 g agarose and add to
50 ml of 1X TAE buffer in a conical flask. Put in oven for about 2 minutes for its
proper mixing. Then immediately add EtBr (not more than 1.5 µL).
3. Now put this gel into the casting tray and allow it to solidify for about 20-25 minutes.
4. When the gel is solidified, carefully remove the combs.
5. Now using micropipette, mix the DNA samples with dye (that consists of
Bromophenol blue and Xylene cyanol).
6. Load the samples mixed with dye into the wells at good speed so that they do not
diffuse into one another.
7. Place the electrode wires appropriately in the electrophoresis unit (that is containing
TE buffer and the casting tray with loaded samples).
8. Start the voltage supply and let the DNA samples migrate towards the anode.
9. After some time, bands can be seen that can either be visualized under a UV
transilluminator or a Gel Doc.

3. RFLP (RESTRICTION FRAGMENT LENGTH

POLYMORPHISM)

This is a technique which was invented by Alec Jeffreys in 1984. This is a simple
technique that involves the restriction digestion of DNA using the restriction enzymes
(restriction endonucleases) and when this DNA is separated into fragments, these are
separated using gel electrophoresis.

This technique exploits the variations that occur in homologous DNA sequences known
as ‘Polymorphisms’, in order to distinguish individuals, populations, or species or to
pinpoint the locations of genes within a sequence.

We know that restriction enzymes are those enzymes that cleave or digest DNA at
specific sites called ‘Recognition sites. Usually the recognition sites are 4-6 base pairs
 (bp) long. Each restriction enzyme targets different nucleotide sequences in a DNA strand
and cleaves at recognition sites. If there is short sequence of GAGC that occurs
repeatedly in the DNA sample, the restriction enzyme that recognizes GAGC sequence
will cut the DNA at every repetition of GAGC pattern. Also, shorter the DNA sequence
recognized, greater the number of fragments generated from digestion.

The human genome is 99.9% same for all the individuals, and the variations arise only
due to 0.1% differences in the genetic makeup.

Working of RFLP:

i. DNA extraction- Firstly, DNA is extracted from blood or saliva or any other sample
and is purified.
ii. DNA fragmentation- Using restriction enzymes, the purified DNA is treated with
restriction enzymes and it is cleaved into fragments.
iii. Gel electrophoresis- Prepare gel (polyacrylamide) for the loading of samples. Connect
with voltmeter and allow the samples to migrate and then visualize using gel doc or
UV transilluminator.
 Two related DNAs will have same recognition sites and the same restriction enzyme
will cut at that site only and hence same kind of bands will be formed (these can be
wild type or variant), this can hence be used in paternity tests.
 Restriction enzyme used- Msp I (cleaves the DNA to an average size of about 5 kb).
This enzyme is anisoschizomer of hpa II.
 FIG. 3 (a): Apparatus for RFLP (showing the migration of samples)

FIG. 3 (b): Gel doc visualization of products

Why the restriction enzyme does not affect the source from which it is
obtained?

As we know that the restriction enzymes are obtained from the bacteria. But an important
question arises that if the function of a restriction enzyme is to cleave DNA then how the
bacterial DNA is protected from the action of restriction enzymes? So, the answer is that the
bacteria prevent the cleavage of their own DNA by the methylation of recognition sites and
hence the bacterial DNA is highly methylated and this prevents the action of restriction
endonucleases on bacterial DNA.

4. PCR (POLYMERASE CHAIN REACTION)

This is a widely used method nowadays in molecular biology, biochemistry and

biotechnological domains. It is used to produce multiple copies of a target or particular DNA
segment. It consists of exponential amplification of copies of DNA sequence in order to
generate thousands to millions of copies of that particular DNA segment.
Key ingredients of PCR:There are two most important ingredients of PCR and these are Taq
polymerase and PCR primers.

 TAQ POLYMERASE- It is a heat stable enzyme which is obtained from the

bacterium Thermus aquaticus.Thisbacterium resides in hot springs and hydrothermal
vents. The heat stability of Taq polymerase at very high temperature (~ 70° C is the
temperature at which it is most stable) makes it ideal choice for PCR as the DNA
polymerase from other bacteria like E. coli are not stable at this temperature.
 PCR PRIMERS: While performing PCR, we add primers of two types and these are
Forward primers and Reverse primers. These are short pieces of ss DNA that range
around 17-30 nucleotides. They are given sequences that bind to the opposite strands
of template DNA by complementary base pairing.
 dNTPs: Deoxynucleoside triphosphates include four basic nucleotides that are: dATP,
dGTP, dCTP, dTTP. These are the basic building blocks of new DNA strands in PCR.
But their concentration higher than the optimum concentration can also inhibit PCR.
 TAQ BUFFER/ PCR BUFFER: This buffer usually contains MgCl2 which is a
cofactor and catalyzer in PCR. Higher concentrations of MgCl2 means the higher
productivity of Taq polymerase. So, the buffer is required for the proper functioning
of the Taq polymerase enzyme. It consists of Tris- HCl, KCl and sometimes MgCl2
(otherwise it has to be added separately).

The process of PCR takes place in the presence of different temperature conditions and in
three steps, which are:

1. DENATURATION: In this step, the double stranded (ds) DNA is converted into
single stranded (ss) DNA. Here the temperature conditions provided are 94°-96° C.
This ss DNA is used as template for next step.
2. ANNEALING: In this step, primers are allowed to bind to the complementary
sequences on the ss- DNA. The temperature in this step can be in the range of 55-65°
C.
3. EXTENSION: In this step, the primers are extended with the help of Taq polymerase
enzyme so as to synthesize complete nucleotide sequence. The temperature here is
72° C.
 FIG. 4: Steps in PCR

After N cycles, PCR generates a 2 N fold increase in the target DNA.

 The purpose of PCR primers is to provide a “free” 3’-OH group to which the
DNA polymerase can add dNTPs.
 Lastly gel electrophoresis can be used to visualize the result of PCR.

5. RT-PCR (REVERSE TRANSCRIPTION POLYMERASE CHAIN

REACTION)

Sometimes when we are unable to get a specific gene sequence or DNA and instead we get
RNA (mRNA) and this RNA acts as template in the process. As we know that RNA is
formed from DNA only by the process of Transcription (according to the Central dogma of
Molecular biology). So, we can also form DNA from RNA by the process called Reverse
Transcription with the use of the enzyme called Reverse transcriptase. This technique is quite
complex because the RNA degrading enzymes, RNases are present ubiquitously. This is the
reason that RNA can degrade easily at the room temperature. So, everything to be used in the
process of RT-PCR is kept in ice at low temperature so as to prevent contamination.

REQUIREMENTS IN RT-PCR: RNA template, enzyme, dNTPs, buffers and thermocyclers.

This technique is also used in combination with qPCR (quantitative PCR or Real Time PCR).
USES: This can be used to qualitatively study gene expression.

 For eg- it can be used to distinguish exons (coding sequences of gene) from introns
(non- coding sequences of gene).
 It can also be used to clinically diagnose various genetic diseases.
 Can also be used for monitoring drug therapy.
 When used with qPCR, this can be used to quantify RNA levels.

FIG. 5: Thermocycler for RT-PCR

CONCLUSION:

As mentioned above, the various techniques discussed are of great importance in research
field. Today we know almost everything about human genome and this became possible only
because we were able to isolate DNA. This nucleic acid is the basis for life and because of
the developments in biology, we have come out with a number of techniques that utilize
DNA and can tell what not. It has become easy to solve the legal disputes regarding paternity
and crime cases. If one gets any sample like hair strand, saliva or blood we can isolate DNA
from the sample. To increase its copies, we can perform PCR that will eventually produce
multiple copies of DNA (in billions). Then, gel electrophoresis can be performed to visualize
the DNA in form of bands. Also, if we are unable to get DNA, rather if we get RNA, we can
perform RT-PCR to obtain DNA (cDNA). RFLP is another important technique that uses
restriction endonucleases to cleave DNA at recognition sites and is very much useful to test
paternity. It is generally followed by blotting and autoradiography.