UNIVERSITY OF KALYANI

MASTER OF SCIENCE SECOND SEMESTER

EXAMINATION 2024

DEPARTMENT OF MICROBIOLOGY

ROLL: 96/MCB NO:

REG NO: SESSION:

Sub: Recombinant DNA Technology

Paper: MB COR 211

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 Index

Serial No. Experiment. Page No.

1. DNA amplification by PCR 3-8
2. Restriction digestion of DNA 9-11

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 DNA AMPLIFICATION BY PCR
Objectives:

 To perform PCR amplification of specific target sequence from genomic DNA.

 To analyze the amplified product by agarose gel electrophoresis.

Principle:

PCR - Polymerase Chain Reaction is an in vitro method of enzymatic synthesis of specific

DNA sequences. This technique was developed by Kary Mullis in 1983. It is a very simple
and inexpensive technique for characterizing, analyzing and synthesizing any specific piece
of DNA or RNA from virtually any living organism (plant, animal, virus or bacteria). It
exploits the natural function of the polymerases, present in all living things, to copy genetic
material or perform "Molecular Photocopying."

PCR consists of three basic steps:

Denaturation: During this step, the two strands of DNA melt open to form single stranded
DNA and all enzymatic reactions stop. This is generally carried out at 92°C -96°C.

Annealing: Annealing of primers to each original strand for new strand synthesis is carried
out between 45°C - 55°C.

Extension: The polymerase adds 2'-deoxynucleoside 5' triphosphates (dNTPs)

complementary to the template at the 3' end of the primers. Since both strands are copied
during PCR, there is an exponential increase in the number of copies of the gene.

These 3 steps are repeated 20-30 times in an automate thermocycler that can heat and cool the
reaction mixture the tube within a very short time. This results in exponent accumulation of
specific DNA fragments, ends of which are defined by 5' ends of the primers. The doubling
of number of DNA strands corresponding to the target sequences allows us to estimate the
amplification associated with each cycle using the formula:

Amplification = 2n, where n = No. of cycles

PCR can amplify a desired DNA sequence of any origin, hundreds of millions of times in
matter of hours, a task that would require days with recombinant technology. It is especially
valuable because the reaction is highly specific, easily automated and very sensitive. It has
thus revolutionized fields like:

 Clinical medicine
 Diagnosis of genetic disorders Forensic sciences
 Evolutionary biology

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Factors affecting Amplification:

I. Reaction components:

Sample Volume:

 Most amplifications are performed at 20, 50, or 100 ∝l volume in 0.2 or 0.5 ml
microfuge tubes.
 Larger volumes do not allow adequate thermal equilibrium of the reaction mixture.

Template DNA:

 Generally nanogram amount of plasmid DNA, or microgram amount of genomic

DNA is used for PCR.
 Higher amounts of template DNA inhibits or results in non-specific amplification.

Primers: PCR reaction needs two primers, a forward and a reverse primer.

 Primers are synthetic oligonucleotides usually ranging from 15 to 30 bases.

 Primers are designed such that at the 3' ends they do not have more than two bases
complimentary to each other, as this results in PRIMER-DIMER formation.
 Guanine-Cytosine content (G+C) content is in the range of 40-60%.
 The melting temperature (Tm) of both forward and reverse primers is usually the
same.
 Low concentrations of primers result in poor yield of the specific product and high
concentrations of primers may result in non-specific amplification. Optimal
concentration of primers is between 0.1-1 μM

Deoxynucleotide-tri-phosphates:

 The final concentration of each dNTP (dATP, dGTP, dCTp and dTTP) in a standard
amplification reaction is 200 μΜ.
 It is important portant to keep the dNTP concentrations above the estimated K of each
dNTP (10 to 15 M) for best base incorporation.

Taq DNA Polymerase buffer:

 The 10X assay buffer contains 100 mM Tris- CI (pH 9.0), 500 mM Potassium
chloride, 15 mM MgCl and 0.1% w/v gelatin.
 Magnesium ions are an essential cofactor required for the activity of Taq DNA
Polymerase. Low concentration of Magnesium will result in no amplification and high
amounts may lead to the production of unwanted products.

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  Taq DNA Polymerase:
 Taq DNA polymerase is a 94 KD (Kilodaltons) thermostable DNA polymerase.
 The Optimal temperature for Taq DNA Polymerase activity is 72°C.
 It lacks 3' to 5' exonuclease activity but has 5' to 3' exonuclease activity and 5' to 3'
polymerase activity.
 For most amplification reactions 1.5 to 2 units of the enzyme recommended as higher
enzyme concentration leads to non-specific amplification.

II. Temperature Cycling: Temperature cycling parameters depend greatly on the template,
primers and amplification apparatus.

Initial Denaturation: Complete denaturation of template DNA at the start of the PCR reaction
is of key importance. Incomplete denaturation results in inefficient utilization of the template
in the first amplification cycle and in a poor yield of the PCR product. It is generally
performed at 95°C for 1-3 minutes, depending on the GC content of the template. If longer
initial denaturation or higher temperature is necessary, then Taq DNA Polymerase can be
added after this step as higher temperature may affect the enzyme stability. Initial
denaturation step is performed only once at the beginning of the reaction.

Denaturation: Subsequent denaturation steps are performed for a shorter time of 30 seconds 1
minute at 94°C for 30 cycles. This is sufficient, since the PCR product synthesized in the first
amplification cycle is significantly shorter than the template DNA and completely denatures
under these conditions. Certain additives like DMSO/glycerol/ formamide are used to
facilitate DNA denaturation depending on the GC content.

Annealing: The optimal annealing temperature is generally 5°C lower than the melting
temperature of primer-template DNA duplex, performed for 30 seconds-1minute. If non-
specific products are obtained in addition to the expected product, the annealing temperature
is optimised by increasing it stepwise by 1-2°C.

Extension: Primer extension, resulting in the synthesis new DNA strand is carried out at 72°
C, which is the optimal temperature for Taq DNA Polymerase activity. The amplification
time is determined by the length of the sequence to be amplified. For every 1 kb target DNA,
one minute tire is recommended. The number of PCR cycles depends on the amount of
template DNA in the reaction mix and on the expected yield of the product.

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Final Extension: As DNA synthesis proceeds, it becomes less efficient as most of the
components get used up. Hence, following the last cycle, the enzyme is allowed to finish any
incomplete synthesis by carrying out a final extension at 72°C for 5-15 minutes

Kit Description: In this kit, reagents are provided to perform PCR using DNA purified from
Serratia marcescens as the template. Two primers, i.e., forward primer and reverse primer are
used to amplify a region of the template that results in a product of size 800 bp. The
amplified product is then analyzed by electrophoresis on a 1.5% agarose gel along with a
marker. The DNA marker is a StepUp™™ 100 bp DNA ladder, with 10 bands ranging from
100 bp to 1 kb.

Duration of Experiment: Approximately 4 hours.

Materials Provided:

The list below provides information about the materials supplied in the kit. The components
should be stored as suggested.

Quantity
Materials 6104400011730 61044000221730 Store
(10 Expts.) (20 Expts.)
10X Assay Buffer 50 μl 0.1 ml -20°C
10 mM dNTP Mix 30 μl 60 μl -20°C
StepupTM 100 bp 50 μl 0.1 ml -20°C
DNA ladder (ready
to use)
Forward Primer 10 μl 20 μl -20°C
Reverse Primer 10 μl 20 μl -20°C
Nuclease Free Water 1 ml 1 ml -20°C
Template DNA 10 μl 20 μl -20°C
Taq DNA 10 μl 20 μl -20°C
Polymerase
Gel Loading Buffer 0.25 ml 0.25 ml -20°C
Agarose 5g 10 g -20°C
50X TAE 20 ml 40 ml RT
Mineral Oil 0.5 ml 1 ml RT
PCR Tubes 10 Nos. 20 Nos. RT

Note: Room Temperature (RT) not to exceed 20°C -25°C

Materials Required:

Equipment: Thermocycler, UV-transilluminator.

Reagents: Distilled Water, Ethidium Bromide.

Other Requirements: Crushed Ice/Genei cooler, Tips, Gloves, Micropipette.

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Note: Read the entire procedure prior to starting the experiment.

 Agarose supplied is sufficient to prepare five 1.5% gels in case of 616104400011730

and ten 1.5% gels in case of 616104400021730. Hence, perform minimum of two
PCR amplifications at a time.
 Agarose gel should be prepared by addition of Ethidium bromide at a concentration of
0.5 µg/ml from a stock of 10 mg/ml (refer Agarose Gel Electrophoresis, page 15).
 Thaw the Template DNA, Forward and Reverse Primer, 10X Assay Buffer on ice
before use.
 Centrifuge the materials provided at 6000 rpm for 5 minutes at room temperature.
 StepUp™™ 100 bp DNA ladder supplied is ready to use and can be loaded directly
onto the agarose gel.

Procedure:

Setting up PCR

1. Add the reagents to the PCR tube in the following order:

Sterile Water 38 μl

10X Assay Buffer 5 μl

10 mM dNTP Mix 3 μl

Template DNA 1 μl

Forward Primer 1 μl

Reverse Primer 1 μl

Taq DNA Polymerase 1 μl

Total Reaction Volume 50 μl

2. Mix the contents gently and layer the reaction mix with 50 µl of mineral oil, to prevent
evaporation.

Note: Mineral oil need not be added if the Thermocycler is equipped with a
heated lid.

PCR Amplification

3. Carry out the amplification in a thermocycler for 30 cycles using the following
reaction conditions:

Initial Denaturation Annealing Extension Final

denaturation Extension
94oC 94oC 48oC 72oC 72oC
1 min 30 sec. 30 sec. 1 min 2 min

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 Restriction digestion of DNA

Aim – To Perform restriction digestion of λDNA with EcoRI and Hind iii and analyze
restriction pattern by agarose get electrophoresis

Principle- To protect, bacteria have developed defense mechanism in term of enzyme

(endonuclease) that chop up any foreign DNA. These molecular scissors found in bacterial
cytoplasm can prove dangerous to cell, so bacteria protect their own DNA by methylating the
adenine or cytosine bases. The methyl groups block the binding of enzymes, but normal
reading and replication of genome are not halted, DNA from attacking bacteriophage will not
have this protective methyl groups and will be destroyed

Together restriction enzymes and its modification, methyl transferase, form a

restriction modification system. Four kind of RM is known. Most characterized enzymes
(about 93%) belong to type II. The type II enzymes recognize specific DNA sequence and
cleave DNA at a fixed location. They act as dimer; each submit recognizing the same 5’ ➡ 3’
sequence complementary DNA strands and hence are said to recognize palindromic
sequences. Before the restriction enzyme cleaves at its specific recognition site and a long
DNA it binds to a site amidst a very large number of non-cognate site. This protein is then
translocated from the initial site to specific site. In the presence of Mg++, the enzyme
undergoes a confirmational change, which kinks the helix and cleaves the DNA producing
‘blunt’ or ‘sticky’ ends

Table:

Enzym Recognition site Fragment site (bp)

e
EcoRI 21226,26104,31749,39163,44972 21226,7421,5804,5643,4878,3530
Hind 23130,25157,27479,36895,37459,37584,4 23130,9416,6557,4961,2322,2027,564
III 4141 ,125

Factors affecting restriction enzyme activity -

Temperature – most digestions with restriction enzymes are carried out at 37°. Digestion
with a restriction enzyme should be carried out at its optimum temperature to achieve
maximum efficiency.

Buffer system – Tris – HCI is the most commonly used buffering agents, most of the
enzymes are active in the PH range 7.0 – 8.0.

Ionic conditions – Mg2++ is an absolute requirement for all restriction endonuclease.

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Methylation of DNA – Methylation of specific adenine or cytosine residues within
recognition sequence of restriction enzyme affects the digestion of DNA.

Requirement:

1. Assay buffer – 12.5 µl in two vials

2. Lamda DNA (substrate) - 10µl.
3. Restriction enzymes, EcoRI and Hind III - 15µl
4. TAE buffer – 4mg in 200 ml water
5. Agarose – 0.5 g in 50ml 1 x TAE
6. Controlled λDNA
7. Ethidium bromide (10mg/ml)
8. Distilled water

Procedure:

First making the restriction recognition mixture

1. Place the enzymes containing vials on ice
2. then add the substrate(λDNA) and 2x assay buffer in that vials and prepare two reaction
mixture in 1.5 ml vials

Reaction-1 Reaction-2
λDNA- 10 µl λDNA- 10 µl
Assay buffer- 12.5 µl Assay buffer- 12.5 µl
EcoRI -1.5 µl Hind III – 1.5 µl

3. Incubate both the vials at 37° for 1hr.

4. After an hour, add 2.5 µl gel loading buffer to stop digestion further.

Second, preparation of 1% Agarose gel:

5. prepare 1x TAE by diluting appropriate amount of 50x TAE buffer (for one experiment,
approx. 200ml of water
6. weight 0.5gm of agarose in 50ml 1x TAE, this gives 1% agarose
7. Heat the agarose until it become transparent and cool it out.
8. Add ethidium bromide to visualize DNA under florescence
9. Place the comb in the casting tray and pour the agarose solution
10. Keep the gel undisturbed at room temperature to solidfty

Electrophoresis:
11. Pour 1x buffer I the gel apparatus stand at 0.5-0.8 cm above the gel surface
12. Gently lift the comb and place the gel tray in the buffer
13. Connect power cord according to convention: Red- anode, black- cathode

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