BIOAXIS DNA RESEARCH CENTRE(P) LIMITED

.Centre For Biological Research

Hyderabad, Andhra Pradesh.

A TRAINING REPORT
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD
OF THE DEGREE OF
BACHELOR OF TECHNOLOGY
(Biotechnology)

SUBMITTED TO LOVELY PROFESSIONAL UNIVERSITY,

JALANDHAR
SUBMITTED BY

Name of Student
J.Durga Srinivas Rao

Registration No.
11306274
30-05-16 to 14-07-16

Lovely Professional University, Jalandhar

PUNJAB

Chapter 1
INTRODUCTION
R DNA TECHNOLOGY

Recombinant DNA molecules arre DNA molecules formed by laboratory methods of genetic
recombination to bring together genetic material from multiple sources, creating sequences that
would not otherwise be found in biological organisms. Recombinant DNA is possible because
DNA molecules from all organisms share the same chemical structure. They differ only in the
nucleotide sequence within that identical overall structure.
In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps:
(1) Choice of host organism and cloning vector,
(2) Preparation of vector DNA,
(3) Preparation of DNA to be cloned,
(4) Creation of recombinant DNA,
(5) Introduction of recombinant DNA into the host organism,
(6) Selection of organisms containing recombinant DNA, and
(7) Screening for clones with desired DNA inserts and biological properties.

Expression of recombinant DNA

Following transplantation into the host organism, the foreign DNA contained within the
recombinant DNA construct may or may not be expressed. That is, the DNA may simply be
replicated without expression, or it may be transcribed and translatedso that a recombinant
protein is produced. Generally speaking, expression of a foreign gene requires restructuring the
gene to include sequences that are required for producing an mRNA molecule that can be used
by the host's translational apparatus (e.g. promoter, translational initiation signal,
and transcriptional terminator). Specific changes to the host organism may be made to improve
expression of the ectopic gene. In addition, changes may be needed to the coding sequences as
well, to optimize translation, make the protein soluble, direct the recombinant protein to the
proper cellular or extracellular location, and stabilize the protein from degradation

Properties of Organisms containing Recombinant DNA

In most cases, organisms containing recombinant DNA have apparently normal phenotypes. That
is, their appearance, behavior and metabolism are usually unchanged, and the only way to

demonstrate the presence of recombinant sequences is to examine the DNA itself, typically using
a polymerase chain reaction (PCR) test.
If the rDNA sequences encode a gene that is expressed, then the presence of RNA and/or protein
products of the recombinant gene can be detected, typically using RT-PCR or western
hybridization methods. Gross phenotypic changes are not the norm, unless the recombinant gene
has been chosen and modified so as to generate biological activity in the host organism.

Applications of R DNA TECHNOLOGY

Recombinant DNA is widely used in biotechnology, medicine and research. Today, recombinant
proteins and other products that result from the use of rDNA technology are found in essentially
every western pharmacy, doctor's or veterinarian's office, medical testing laboratory, and
biological research laboratory. In addition, organisms that have been manipulated using
recombinant DNA technology, as well as products derived from those organisms, have found
their way into many farms, supermarkets, home medicine cabinets, and even pet shops, such as
those that sell GloFish and other genetically modified animals.

EXAMPLES
Recombinant human insulin
Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle)
for the treatment of insulin-dependent diabetes. A variety of different recombinant
insulin preparations are in widespread use. Recombinant insulin is synthesized by
inserting the human insulin gene into E. coli, or yeast (saccharomyces cerevisiae) which
then produces insulin for human use.

Recombinant hepatitis B vaccine

Hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine,
which contains a form of the hepatitis B virus surface antigen that is produced in yeast
cells. The development of the recombinant subunit vaccine was an important and
necessary development because hepatitis B virus, unlike other common viruses such
aspolio virus, cannot be grown in vitro.
Golden rice
A recombinant variety of rice that has been engineered to express the enzymes
responsible for -carotene biosynthesis.This variety of rice holds substantial promise for
reducing the incidence of vitamin A deficiency in the world's population.

Chapter 2
Isolation of DNA from various tissues
2.1.1. DNA Isolation from Banana
Step 1 Prepare Hyper saline solution
Step 2 Take a wet tissue of banana and grind it with hyper saline solution
Step 3 keep it at room temperature

Step 4 Centrifuge at 10,000 rpm for 10 minutes

Step 5 Take supernatant and add equal volume of isopropyl alcohol

Composition :
Hyper saline solution : 1.2 gm in 100 ml
Principle:
Hyper saline solution disturbs the osmotic stability of the cells and bursts the cells releasing
DNA.
Isopropyl alcohol decreases dielectric constant there by brings DNA molecules together to
precipitate.

2.1.2.Isolation of DNA from leaf material

Step 1. take a leaf sample and grind it in the presence of extracrion buffer
Step 2. incubate at 60 65 degrees for for 1 hour. Gently vortex every 10 mints
Step 3. Allow it to come to room temperature and add 5 ml of PCI
( phenol:choloform:isoamylalcohol) in the ratio 25 :24:1
Step 4. incubate at 4 degree overnight
Step 6 centrifuge at 12000 rpm for 1o minutes and take the pellet ,dissolve in TE buffer

composition:
Extraction buffer (w/v)
3% CTAB (0.3 gm in 10 ml)
100mM tris Hcl
20 mM EDTA
1.4 M Nacl
0.2 %(v/v) beta mercapto ethanol
0.2 % pvp (poly vinyl propilidone)
Principle :
All plant DNA extraction comprise of the basic steps of disruption of the cell wall, cell
membrane and nuclear membrane to release the DNA into solution followed by precipitation of
DNA while ensuring removal of the contaminating biomolecules such as the proteins,
polysaccharides, lipids, phenols and other secondary metabolites. This is brought about by
disruption of the tissue in a mortar and pestle aided by liquid nitrogen and the various
components of the homogenization or extraction buffer followed the precipitating and
purification methods employed. Since DNA can be extracted from various types of tissues such
as seedlings, leaves, cotyledons, seeds, endosperm, tissue culture callus, roots etc., the tissue
type along with the concentration of DNA finally required determine the methodology of DNA
extraction to be followed by the experimenter.

EDTA : Ethelene diamine terta acetic acid is a chelating agent which chelate all ions present in
the solution.
Tris Hcl: Tris being a base and Hcl being a acid forms a buffer which simulates the buffer
condition in living system so as to maitain P H value.
CTAB : The best detergent to use during the extraction/isolation of highly polymerized DNA
from plant material. This detergent simultaneously solubilizes the plant cell wall and
lipid membranes of internal organelles and denatures proteins (enzymes). Thus, the
DNA is not hydrolyzed during the isolation process and as long as vortexing or
vigorous shaking are avoided highly polymerized (i.e., very high intact) genomic DNA
is obtained.
Phenol chloroform : Nucleic acid solutions commonly contain undesirable contaminants that
are chiefly made of proteins. A classic method of purifying is phenol chloroform extraction by
which the the nucleic acid solution is extracted by successively washing with a volume of
phenol(pH 8.0); a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and chloroform:
isoamyl alcohol ( 24:1). Centrifugation is performed intermittently and the upper aqueous phase
is transferred to a new tube while avoiding the interphase. The contaminants are denatured and
and accumulate in the organic phase or in the marginal layer between the two phases and the
nucleic acids are preserved in the aqueous phase.
Precipitation :Alcohol precipitation is the most commonly used method for nucleic acid
precipitation. This requires diluting the nucleic acid with a monovalent salt , adding alcohol to it
and mixing gently. The nucleic acid precipitated spontaneously and can be pelleted by
centrifugation. The salts and alcohol remnants are removed by washing with 70% alcohol. The
most commonly used salts include sodium acetate pH 5.2(final volume 0.3M), sodium chloride
(final concentration 0.2M), ammonium acetate (2- 2.5M), lithium chloride (0.8M) and potassium
chloride. Ethanol (twice the volume) or isopropanol ( two thirds volume) are the standard
alcohols used for nucleic acid precipitation.

2.1.3. BUCCAL DNA ISOLATION

Step 1. Swish 2 ml of hyper saline solution around in your mouth for 30 sec
Step 2. Centrifuge at 4000 rpm for 5 min
Step 3. Pouroff supernatant without disturbing pellet
Step 4. Add 1ml of lysis buffer and mix by carefully shaking it
Step 5. Incubate at 65 degrees
Step 6. Add 500 ul 5M Nacl to the dilution and mix gently for 1 min
Step 7. Spin at 4000 rpm for 10 min
Step 8. Transfer 500 ul of supernatant into appendrof and add 1 ml of cold isopropanol
Step 9. Mixwell by inverting the tubes
Step 10. Incubate at 4 degrees overnight
Step 11. Spin at 4000 rpm for 10 min
Step 12. Add 1ml of 70% ETOH to pellet
Step 13. Invert the tube, spin at 10000 rom for 5 min
Step 14. Air dry the pellet
Step 15. Dissolve pellet in 100 ul buffer.

Principle:
Hyper saline solution disturbs the osmotic stability of the cells and bursts the cells releasing
DNA.
Isopropyl alcohol decreases dielectric constant there by brings DNA molecules together to
precipitate

2.1.4. BLOOD DNA ISOLATION

Step 1. Take 1ml of blood(EDTA) and add 3 ml of solution A
Step 2. Shake it slowly for 10 min
Step 3. Incubate at 37 degree
Step 4. Centrifuge for 5 min at 5000 rpm
Step 5. Take pellet and add 2 vol of solution B .shake it.
Step 6. Incubate at 37 degrees for 30 min
Step 7. Add 650 ul of 3M sodium acetate
Step 8. Incubate at 65 degree for 20 min
Step 9. Add 650 ul of icecold choloform
Step 10. Shake it for 60 min at 4000 rpm for 10 min
Step 11. Take supernatant and equal vol. Of icecold isopropanol
Step 12. Incubate at 20 degree for 30 min and centrifuge at 12000 rpm
Step 13. Wash the pellet at 70% ETOH
Step 14. Discard supernatant air dry the pellet
Step 15. Dissolve DNA in 50 -100 ul TE buffer.
Composition:
Solution A (100 ml)

Solution B(100 ml)

1 M tris Hcl pH 7.8

1 M tris Hcl (pH 7.8)

Sucrose 10.9 grams

0.5 M EDTA (pH 8.0)

Mgcl 2 47 mg
0.876grams

Nacl

EDTA : Ethelene diamine terta acetic acid is a chelating agent which chelate all ions present
in
the solution.

Tris Hcl: Tris being a base and Hcl being a acid forms a buffer which simulates the buffer
condition in living system so as to maitain P H value.
2.1.5. YEAST GENOMIC DNA ISOLATION
Step 1. Take 1.5 ml of overnight culture of yeast cells to a microfuge tube
Step 2. Centrifuge at maximum speed for 3 min
Step 3. Remove the supernatant and resuspend the pellet in 50 ul of STE buffer.
Step 4. Add 20 ul of TE buffer (pH 7.6)
Step 5. Add 60 ul of phenol : choloform : isoamylalcohol (25:24:1) and vortex for 1 min
Step 6. Centrifuge at max. Speed for 5 min
Step 7. Transfer upper aqueous phase to a fresh tube
Step 8. Precipitate DNA to 100 % ETOH for 15 min at 0 degree
Step 9. Pellet DNA by centrifuge at maximum speed for 10 minutes at 4
degrees
Step 10. Rinse the pellet at 100 ul of 70 % ETOH
Step 11. Centrifuge at maximum speed for 1 min
Step 12. Air dry the pellet for 15 min and redissolve in 40 ul TE buffer
COMPOSITION:
STE buffer pH 7.6
0.2 M Tris Hcl
0.5 M Nacl
0.01 M EDTA NA 2
EDTA : Ethelene diamine terta acetic acid is a chelating agent which chelate all ions present
in
the solution.

10

Tris Hcl: Tris being a base and Hcl being a acid forms a buffer which simulates the buffer
condition in living system so as to maitain P H value.

11

2.1.6. PLASMID ISOLATION

A plasmid is a small DNA molecule within a cell that is physically separated from a
chromosomal DNA and can replicate independently. They are most commonly found in bacteria
as small, circular, double-stranded DNA molecules.

Step 1. Prepare 50 ml of LB broth,sterilize and inocualte Ecoli strain with plasmid into it.
Step 2. Take 1.5 ml of overnight culture and centrifuge at 6000 rpm for 10 min.
Step 3. Take the pellet and add 150 ul of solution 1 and genntly vortex it for 1min.
Step 4. keep it in icecold condition for 10 min
Step 5. Add 100 ul of solution 2
Step 6. Gently vortex till white,viscous layer appears. Keep it in icecold condition for 10 min.
Step 7. Add 200 ul of solution 3
Step 8. Gently vortex for 2 min. Keep in icecold condition Centrifuge at 8000 rpm for 10 min.
Step 9. Take supernatant and add equal vol. of isopropanol and keep it in icecold condition for
30 min.
Step 10.Centrifuge at 12000 rpm for 10 min.Take the pellet,dry it and dissolve in 50 ul TE
buffer.
Composition
Solution 1

solution 2

solution 3

50 mM Tris Hcl

0.2 N NaOH

20 mM EDTA

1.5 % SDS

3 M Na acetate

15 % Glucose
Principle :
Alkaline lysis is a method used to isolate plasmid DNA or other cell components such as
proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown,
and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl
sulfate (SDS) and a strong base sodium hydroxide.

12

The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the
proteins which are involved in maintaining the structure of the cell membrane. Through a series
of steps involving agitation, precipitation, centrifugation, and the removal of supernatant,
cellular debris is removed and the plasmid is isolated and purified.
Sodium dodecyl sulfate: (SDS) (C12H25SO4Na) or sodium lauryl sulfate (SLS) is an anionic
surfactant. It is a molecule having a tail of 12 carbon atoms, attached to a sulfate group. This
sulfate group provide the amphiphilic properties (required for a detergent) to the molecule.
EDTA : Ethelene diamine terta acetic acid is a chelating agent which chelate all ions present in
the solution.
Tris Hcl: Tris being a base and Hcl being a acid forms a buffer which simulates the buffer
condition in living system so as to maitain P H value.
Na acetate : sodium acetate is the used to bring P H of the solution to normal so that plasmid
renaturation occurs leaving long genomic DNA denatured.
NaOH : NaOH is a strong Base which creates alakine condition for denaturation of genomic
DNA .

2.2. Restriction Digestion

Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at
specific sequences. There are hundreds of different restriction enzymes to target a wide variety
of recognition sequences.
Example :
Action of Eco RI restriction enzyme

13

PROTOCOL:
Step 1. Take 7 ul of lambda DNA and add 2 ul of ECO RI
Step 2. Add 2.5 ul of 10 X assay buffer and make up the vol. To 25 ul by adding D H20
Step 3. Incubate at 37 degree for 1 hr
Step 4. Add 2 ul of EDTA solution
Step 5. Run the samples on agarose gel

Ligation:
Step 1. Take 7 ul of restriction digested lambda DNA and add 2 ul of Ty DNA ligase
Step 2. Make up the volume to 25 ul by adding DH20
Step 3. Incubate at 17 to 22 degree for 2 hrs

Principle:
14

EcoRI is an endonuclease enzyme isolated from strains of E.Coli, and is part of the restriction
modification system.
The restriction site for Eco RI is
G A A T T C
C T T A A G
Restriction enzymes such as EcoRI are used in a wide variety of molecular genetics techniques
including cloning DNA screening and deleting sections of DNA in vitro. Restriction enzymes like
EcoRI that generate sticky ends of DNA are often used to cut DNA prior toligation, as the sticky
ends make the ligation reaction more efficient.
STAR ACTIVITY :
Depending on the conditions present in the reaction. Conditions that can induce star activity
when using EcoRI include low salt concentration, high glycerol concentration, excessive
amounts of enzyme present in the reaction, high pH and contamination with certain organic
solvents.

Use of EDTA :
EDTA being a chelating agent it chelates all ions present in the solution so that all ions required
for function of enzymes as cofactors are tightly packed and make ions unavailable for action of
restriction enzuymes further on DNA.

2.3. Transformation
Sensitive Strain preparation:
Step 1. Inoculate E.Coli Dh5 alpha strain and incubate overnight
Step 2. Take 1.5 ml culture and centrifuge at 6000 rpm for 10 min. Step
3. Take the pellet and add 1 ml of 80 mM Mgcl 2 and 20 mM cacl2
Step 4. Keep it in icecold condition for 10 min.

15

Step 5. centrifuge at 6000 rpm for 10 min Step 6.

Take the pellet & add 1 ml of 100 mM cacl2
Step 7. keep it in icecold condition for 10 min.
Step 8. centrifuge at 6000 rpm.
Transformation step :
Step 1. Take the pellet and add 2 ul of plasmid PUC 18
Step 2. Keep it in icecold condition for 10 min.
Step 3. Incubate at 42 degree for 90 sec to 2 minutes
Step 4. Keep it in icecold condition
Step 5. Take the culture and add 1 ml of sterile broth
Step 6. keep it at 37 degree for 1 hr.
Step 7. Inoculate the incubated culture suspension onto solidified LB Agar plate
Step 8. Observe for transformed colonies after overnight incubation.
RESULT

2.4. Agarose Gel electrophoresis :

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled
bundles that are aggregated into three-dimensional structures with channels and pores through
which biomolecules can pass.
Agarose gel has large pore size and good gel strength that made it particularly suitable as an
anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore
size of a 1% gel has been estimated from 100 nm to 200-500 nm.
Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range
of separation, and is therefore used for DNA fragments of usually 50-20,000 bp in size. The limit
16

of resolution for standard agarose gel electrophoresis is around 750 kb, but resolution of over 6
Mb is possible with pulsed field gel electrophoresis (PFGE).
Staining :
DNA as well as RNA are normally visualized by staining with ethidium bromide, which
intercalates into the major grooves of the DNA and fluoresces under UV light. The ethidium
bromide may be added to the agarose solution before it gels, or the DNA gel may be stained later
after electrophoresis. Destaining of the gel is not necessary but may produce better images. Other
methods of staining are available; examples are SYBR Green, GelRed, methylene blue, brilliant
cresyl blue, Nile blue sulphate, and crystal violet. SYBR Green, GelRed and other similar
commercial products are sold as safer alternatives to ethidium bromide as it has been shown to
be mutagenic in Ames test, although the carcinogenicity of ethidium bromide has not actually
been established. SYBR Green requires the use of a blue-light transilluminator. DNA stained
with crystal violet can be viewed under natural light without the use of a UV transilluminator
which is an advantage, however it may not produce a strong band.

PROTOCOL:
Step 1 weigh 0.5 gm of agarose powder
Step 2 prepare 50 ml of 1x TE buffer
Step 3 dissolve the agarose in TE buffer by heating.
Step 4 after coooling for a while add Ethidium bromide 20 ul in 50 ml agarose.
Step 5 now cast the gel in gel casting tray and leave it until it is solidified
Step 6 after gel is solidified add dip the gel in buffer tank contating 1 x TE buffer.
Step 7 add the samples to the gel with gel loading dye
Step 8 Run the gel for 1 hr by observing the bands of bromophenol blue
Step 9 take the gel off the tank and observe it under UV
Composition :
Gel loading dye
Bromo phenol blue 3%

17

Glycerol

60%

Distiller water

37%

Analysing the gel :

Using the DNA ladder in the first lane as a guide, you can interpret the bands that you get in your
sample lanes to determine the resulting DNA bands.
Ladder is a group of DNA molecules for various size which on runnig in agarose
gel resolves into various bands based on the molecular weight and one can easily measure what
is the molecular weight of our DNA .
Staining the DNA by FAST BLAST DNA STAIN :
even though ethidium bromide is cheap, has good resolution and is relatively easy to use, it can
be extremely dangerous in the hands of someone that is not ready to accept the responsibility to
handle such a dangerous chemical. It can easily be absorbed through the skin, eyes, or
respiratory tract where it will bind tightly to DNA, acting as a mutinagen. Spills in a lab are also
very costly to clean up as well as disposal of ethidium bromide waste.
Fast Blast a 5:1 mixture with water was made and gel was soaked for 2-3 minutes. Remainder
of stain may be stored in a bottle and used for a total of 8 times before disposal. Gel was then
rinsed with warm tap water, changing tap water as it darkens as agitating for 30 minutes to
overnight.
Fast Blast- Use 33l per 50 ml agrose. As well as 200l Fast Blast /300 ml buffer. If necessary
destain after.
Fast Blast offers a great versatility as it can be used as a 5x concentration that may be used up to
8 times before disgaurding and distained or stained overnight at 1x concentration and visualized
without distaining. Destaining did take several rinses in warm water and worked even better
destaining overnight. Gels may be dried and stapled into lab books if so desired, though proper
drying requires a gel dryer.
Its resolution was comparable to that of ethidium bromide and its cost was 22 cents per gel. If
destaining is not seen as a problem, this may be the easiest of non-fluorescent stains.
Since a UV trans illumonator is not required, this stain has some definite pluses.
Step 1 prepare 100 X sast blast DNA stain
Step 2 stain gel for 2-3 mins by adding the gel to buffer containing fast blast DNA stain
Step 3 after running rinse the gel

18

Step 4 wash gel by luke warm water there by removing stain

Step 5 wash the gel once again by adding destaining buffer
Step 6 analyse the results.

Results obtained :

19

Agarose gel

Fast blast DNA stain :

2.5. POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single
copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands
to millions of copies of a particular DNA sequence.
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA
fragments) containing sequences complementary to the target region along with a DNA
polymerase, which the method is named after, are key components to enable selective and
repeated amplification. As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is exponentially
amplified. PCR can be extensively modified to perform a wide array ofgenetic manipulations.

20

Steps for PCR

Initial denaturtion

Temperature

Time

95 degrees

1- 3 min.

Step 1 Denaturation

95 degrees

60 -90 sec

Step 2 Annealing

60 degrees

30 sec

Step 3 Extension

72 degrees

60 sec

72 degrees

2-4 min.

Final Extension

Table

PCR Steps

Types of PCR :
Multiplex PCR uses a number of pairs of primers to allow analysis of a number of fragments
in a single sample
Nested PCR after an initial 30-35 cycles of PCR, an additional round of PCR uses new
primers nested within the original primers, making the process more sensitive because it
reduces the risk of unwanted products from primers binding to incorrect regions
qPCR in quantitative PCR (also known as real time PCR, RT PCR or qRT-PCR), the DNA or
RNA molecules are tagged using fluorescent probes, so that the concentration of amplified
products can be monitored and quantified in real-time by tracking the level of fluorescence
Reverse transcriptase PCR confusingly, also known as RT PCR creates cDNA
(complementary DNA) by reverse transcribing RNA to DNA using reverse transcriptase
Conventional PCR the basic PCR process, which produces up to a billion copies of a DNA or
RNA strand; the results are only seen at the end of the process
Asymmetric PCR amplifies just one strand of the target DNA
Allele-specific PCR this uses allele-specific primers that are designed to match a mutation

21

Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA
sequences by performing PCR on a pool of long oligonucleotides with short overlapping
segments. The oligonucleotides alternate between sense and antisense directions, and the
overlapping segments determine the order of the PCR fragments, thereby selectively producing
the final long DNA product.

Components of PCR:
1.DNA template that contains the DNA region (target) to amplify
2. Two primers that are complementary to the 3' (three prime) ends of each of the sense and
anti-sense strand of the DNA target
3. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C
4. Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide triphosphates";

nucleotides containing triphosphate groups), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand
5. Buffer solution, providing a suitable chemical environment for optimum activity and stability

of the DNA polymerase

22

6. Bivalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be

used for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error
rate during DNA synthesis
7.Monovalent cation potassium ions

Patent :
The PCR technique was developed and patented by Kary Mullis and assigned to Cetus
Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme
was also covered by patents.
Taq polymerase :

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an
enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase
enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by
using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers),
which are required for initiation of DNA synthesis. The vast majority of PCR methods use
thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of
temperature steps.

Gene amplification :
The gene of interest that was supposed to be amplified is
ASPM Abnormal spindle like microcephaly homolog protein
Abnormal spindle protein homolog or Asp homolog is a protein that in humans is encoded by the
ASPM gene. ASPM is located on chromosome 1, band q31 (1q31). Defective forms of the
ASPM gene are associated with autosomal recessive primary microcephaly.
Template DNA

5 ul

Reaction buffer

1 ul

10mM dNTPs

2 ul

23

10 uM forward primer

1ul

10 uM reverse primer

1ul

Taq DNA polymerase

1ul

Nuclease free water

10 ul

Thermocycling conditions for a routine PCR:

STEP

TEMP

Initial Denaturation

95C

30 Cycles

95C
45-68C
68C

Final Extension

68C

Hold

Table

4-10C

PCR Steps

Results obtained :

24

TIME
30 seconds

15-30 seconds
15-60 seconds
1 minute/kb
5 minutes

Figure: PCR

2.6. PRIMER DESIGNING

The important design considerations described below are a key to specific amplification with
high yield. The preferred values indicated are built into all our products by default.
Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp.
This length is long enough for adequate specificity and short enough for primers to bind easily to
the template at the annealing temperature.
Primer Melting Temperature: Primer Melting Temperature (Tm) by definition is the
temperature at which one half of the DNA duplex will dissociate to become single stranded and
indicates the duplex stability. Primers with melting temperatures in the range of 52-58 oC
generally produce the best results. Primers with melting temperatures above 65oC have a
tendency for secondary annealing. The GC content of the sequence gives a fair indication of the
primer Tm. All our products calculate it using the nearest neighbor thermodynamic theory,
accepted as a much superior method for estimating it, which is considered the most recent and
best available.

Tm calculation
Melting temperature in Kelvin :
Tm(K)={H/ S + R ln(C)}
Melting Temperature in temperature :
Tm(oC) = {H/ S + R ln(C)} - 273.15
H (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by
substances. H is the change in Enthalpy. In the above formula the H is obtained by adding up
all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.
S (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. S is change in
Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each
nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor
parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the
default condition used for all calculations.

25

S (salt correction) = S (1M NaCl )+ 0.368 x N x ln([Na+])

Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.
[Na+] calculation:
[Na+] = Monovalent ion concentration +4 x free Mg2+.
Primer Annealing Temperature: The primer melting temperature is the estimate of the
DNADNA hybrid stability and critical in determining the annealing temperature. Too high Ta
will produce insufficient primer-template hybridization resulting in low PCR product yield. Too
low Ta may possibly lead to non-specific products caused by a high number of base pair
mismatches,.
Mismatch tolerance is found to have the strongest influence on PCR specificity
Ta = 0.3 x Tm(primer) + 0.7 Tm (product) 14.9
where,
Tm(primer) = Melting Temperature of the primers
Tm(product) = Melting temperature of the product

GC Content: The GC content (the number of G's and C's in the primer as a percentage of the
total bases) of primer should be 40-60%.
GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers
(GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C
bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
Primer Secondary Structures: Presence of the primer secondary structures produced by
intermolecular or intramolecular interactions can lead to poor or no yield of the product. They
adversely affect primer template annealing and thus the amplification. They greatly reduce the
availability of primers to the reaction.
Hairpins: It is formed by intramolecular interaction within the primer and should be avoided.
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Optimally a 3' end hairpin with a G of -2 kcal/mol and an internal hairpin with a G of -3
kcal/mol is tolerated generally
G definition: The Gibbs Free Energy G is the measure of the amount of work that can be
extracted from a process operating at a constant pressure. It is the measure of the spontaneity of
the reaction. The stability of hairpin is commonly represented by its G value, the energy
required to break the secondary structure. Larger negative value for G indicates stable,
undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.
G = H TS
ii) Self Dimer: A primer self-dimer is formed by intermolecular interactions between the two
(same sense) primers, where the primer is homologous to itself. Generally a large amount of
primers are used in PCR compared to the amount of target gene. When primers form
intermolecular dimers much more readily than hybridizing to target DNA, they reduce the
product yield. Optimally a 3' end self dimer with a G of -5 kcal/mol and an internal self dimer
with a G of -6 kcal/mol is tolerated generally. iii) Cross Dimer: Primer cross dimers are
formed by intermolecular interaction between sense and antisense primers, where they are
homologous. Optimally a 3' end cross dimer with a G of -5 kcal/mol and an internal cross
dimer with a G of -6 kcal/mol is tolerated generally.

Repeats: A repeat is a di-nucleotide occurring many times consecutively and should be avoided
because they can misprime. For example: ATATATAT. A maximum number of di-nucleotide
repeats acceptable in an oligo is 4 di-nucleotides.
Runs: Primers with long runs of a single base should generally be avoided as they can misprime.
For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A maximum
number of runs accepted is 4bp.
3' End Stability: It is the maximum G value of the five bases from the 3' end. An unstable 3'
end (less negative G) will result in less false priming.
Avoid Template Secondary Structure: A single stranded Nucleic acid sequences is highly
unstable and fold into conformations (secondary structures). The stability of these template
secondary structures depends largely on their free energy and melting temperatures(Tm).
Consideration of template secondary structures is important in designing primers, especially in
qPCR. If primers are designed on a secondary structures which is stable even above the

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annealing temperatures, the primers are unable to bind to the template and the yield of PCR
product is significantly affected.
Hence, it is important to design primers in the regions of the templates that do not form stable
secondary structures during the PCR reaction. Our products determine the secondary structures
of the template and design primers avoiding them.

Avoid Cross Homology: To improve specificity of the primers it is necessary to avoid regions
of homology. Primers designed for a sequence must not amplify other genes in the mixture.
Commonly, primers are designed and then BLASTed to test the specificity. Our products offer a
better alternative. You can avoid regions of cross homology while designing primers. You can
BLAST the templates against the appropriate non-redundant database and the software will
interpret the results. It will identify regions significant cross homologies in each template and
avoid them during primer search

PARAMETERS FOR PRIMER PAIR DESIGN

Amplicon Length: The amplicon length is dictated by the experimental goals. For qPCR, the
target length is closer to 100 bp and for standard PCR, it is near 500 bp. If you know the
positions of each primer with respect to the template, the product is calculated as: Product length
= (Position of antisense primer-Position of sense primer) + 1.
Product Position: Primer can be located near the 5' end, the 3' end or any where within specified
length. Generally, the sequence close to the 3' end is known with greater confidence and hence
preferred most frequently.
Tm of Product: Melting Temperature (Tm) is the temperature at which one half of the DNA
duplex will dissociate and become single stranded. The stability of the primer-template DNA
duplex can be measured by the melting temperature (Tm)
Optimum Annealing Temperature (Ta Opt): The formula of Rychlik is most respected. Our
products use this formula to calculate it and thousands of our customers have reported good
results using it for the annealing step of the PCR cycle. It usually results in good PCR product
yield with minimum false product production.

TA Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9

where

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Tm of primer is the melting temperature of the less stable primer-template pair

Tm of product is the melting temperature of the PCR product.
Primer Pair Tm Mismatch Calculation: The two primers of a primer pair should have closely
matched melting temperatures for maximizing PCR product yield. The difference of 5oC or more
can lead no amplification

Primer-Primer interactions
When designing primers, it is important to have a minimum of intramolecular or
intermolecular homology. This would result in either hairpins or primer dimerization.
If a primer has a region of self-homology, snap back or partially double-stranded
structures can occur which will interfere with annealing to the template. Usually
intraprimer homologies of 3 bp or more should be avoided.

Ideal primer designed having G=-1.6 kcal/mol

Reverse Transcriptase PCR :

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In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a
reverse transcriptase. The cDNA is then used as a template for exponential amplification using
PCR. QT-NASBA is currently the most sensitive method of RNA detection available.The use of
RT-PCR for the detection of RNA transcript has revolutionalized the study of gene expression in
the following important ways:

Made it theoretically possible to detect the transcripts of practically any gene

Enabled sample amplification and eliminated the need for abundant starting material that one
faces when using northern blot analysis
Provided tolerance for RNA degradation as long as the RNA spanning the primer is intact
Genetic Disease Diagnosis
RT-PCR can be used to diagnose genetic disease such as LeschNyhan syndrome. This genetic
disease is caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal uric
acid urinary stone and symptoms similar to gout.[6] Analyzing a pregnant mother and a fetus for
mRNA expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will
likely to develop LeschNyhan syndrome.
CHALLENGES:
Despite its major advantages, RT-PCR is not without drawbacks. The exponential growth of the
reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces
inaccurate end point quantification due to the difficulty in maintaining linearity.

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CHAPTER 3

Conclusion and Future scope

Recombinant DNA research is a challenging field, but it holds great promise for the future. In
the future, rDNA technologies will play a key role in preventing genetic diseases, producing
targeted medicines, and providing patients with less toxic pharmaceuticals. It will also impact
agriculture and livestock as researchers find ways to optimize the genetic codes of plants and
animals to resist disease.

In some cases, recombinant DNA can have deleterious effects even if it is not expressed. One
mechanism by which this happens is insertional inactivation, in which the rDNA becomes
inserted into a host cell's gene. In some cases, researchers use this phenomenon to "knock out"
genes to determine their biological function and importance.Another mechanism by which rDNA
insertion into chromosomal DNA can affect gene expression is by inappropriate activation of
previously unexpressed host cell genes.

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REFERENCES

[1] http://onlinelibrary.wiley.com/doi/10.1111/j.
[2] http://biology.kenyon.edu/courses/biol114/Chap08/Chapter_08a.html
[3] http://www.pharmacopeia.cn/v29240/usp29nf24s0_c1045s1.html
[4] www.tactindia.org/future.html

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