An Assignment

on
Polymerase chain reaction(PCR) Concept
and it’s Application in Agriculture

Group No. 3

 ASH1714036M  BKH1714043F
 ASH1714038M  ASH1714044M
 ASH1714039M  ASH1714045M
 BKH1714042F  ASH1714046M
History of polymerase Chain reaction (PCR)
 1983: Dr.Kary Mullis developed (PCR).
 1985: First Publication of PCR by Cetus Corporation appears in science.
 1986: Purified taq Polymerase is first used in PCR.
 1988: Perkin Elmer introduces the automated thermal cycler.
 1989: Science declares taq polymerase " molecule of the year ″
 1990: amplification and detection of specific DNA sequence using a fluoresent
DNA binding dye, laying the fundation for future ''real-time'' or ''kinetic'' PCR.
 1993: Dr.Cary Mullis shares noble prize in chemistry for coceiving PCR
technology.
 1999: Dynal launches DRB-36 HLA-typing kit for tissue typing.
 2003:HIV-1 MONITOR test,version 1.5 product family.
Concept of Polymerase Chain Reaction(PCR):

 Polymerase Chain Reaction (PCR) is a technique used in molecular biology to

amplify a single copy or a few copies of a segment of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA
sequence.

 Developed in 1983 by Kary Mullis, PCR is now a common technique used in

clinical and research laboratories for a broad variety of applications.

 In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.
Types of PCR:
Multiplex PCR:
 Uses a number of primers to multiply multiple fragments in a single.

Nested PCR:
 After the initial 25-35 PCR cycles, an additional PCR is conducted using new primers
“nested” within the original primers, which reduces the risk of unwanted products.
Real-Time PCR (quantitative PCR or qPCR):
 In which DNA molecules are tagged using fluorescent dye, which is used to monitor and
quantify PCR products in real-time.
Reverse-Transcriptase (RT–PCR):
 Creates complementary DNA (cDNA) by reverse transcribing RNA to DNA using reverse
transcriptase.
Assembly PCR:
 Longer DNA fragments are amplified by using overlapping primers.

Asymmetric PCR:
 Only one strand of the target DNA is amplified.
Long-range PCR:
 Longer ranges of DNA are formed by using a mixture of polymerases.

Hot Start PCR:

 In which heat is used to de-naturate antibodies that are used to inactivate Taq polymerase.

In situ PCR:
 PCR that takes place in cells, or in fixed tissue on a slide.
Touchdown PCR:
 This type of PCR is used to optimize yield of amplified product at different annealing
temperature.
Basic Requirements and Equipments of Polymerase Chain Reaction (PCR)

Basic requirements:
1) DNA sequence of target region must be known.

2) Primers:
 Typically 20-30 bases in size.These can be prepared using a DNA synthesizer.

3) Thermo-stable DNA polymerase:

 eg. Taq polymerase which is not inactivated by heating to 95c

4) DNA thermal cycler:

 Machine which can be programmed to carry out heating and cooling of samples
over a number of cycles.
Equipments:
 Autoclaves and  Dry and wet baths Chemical Components:
sterilizers  UV transilluminators  Magnesium chloride: 5-
 Clean room tables,  Vortexers/Vortex 2.5mM
gloves, workstations mixer  Buffer: pH 8.3-8.8
and finger cots
 Vibration isolation  dNTPs: 20-200µM
 Pipettors
workstations
 Shakers  Primers: 0.1-0.5µM
 Hand washers and
 Dispensers  DNA polymerase: 1-2.5
dryers
units
 Consumable kits and  Analytical
 Target DNA: ≤ 1µg
tube instruments
 Incubators
 Gel Doc systems
 Labware washers
Steps in PCR:
1.Initialization:
 Heating the reaction to a temperature of 94-96c
for 1-9minutes.It is done in the very begining of
pcr reaction.
2.Denaturation:
 The reaction mixture is heated to 94c for
1minutes. Double stranded DNA melts-----single
stranded DNApicture
3.Annealing:
 Heated to 50-65c for 20-40seconds(dependant on
the melting temperature of the expected duplex).
Primers binds to their complementary sequences.
4.Extension:
 Temperature :~72c for. 5- 3minutes.
5.Final elongation:
 70-74c for 5-15 minutes. To ensure that remaining single stranded DNA is fully extended.
6.Final hold:
 4-15c for an indefinite time.Short-term storage of the reaction.
Procedure of Polymerase chain reaction(PCR):

TaqMan® system (used for molecular feasibility testing):

 Turn on the molecular biology cabinet and leave UV light on for 10 minutes;
 Inside the cabinet, dilute the stock of primers and probe in nucleases free water (eg RF) or
TE buffer (see information from this solution below) at 10 μM concentration in
RNAse/DNAse-free PCR microtubes (eppendorf or similar). Typically, the stock of primers
at an initial 100 μM concentration needs to be diluted 1:10.
 Prepare the Mix containing the primers by adding:
 0.2 μL Primer Reverse (10 μM)
 0.2 μL Primer Sense (10 μM)
 0.2 μL Probe (10 μM)
 5.0 μL master mix
 2.4 μL Water RF
 Add 8μL of the reaction MIX per well to the PCR plate (96-well or 384-well plate)
  Outside of the cabinet, add 2 μL of cDNA or DNA (cDNA or DNA concentration 5 ng / μL)
per reaction. In this way the reaction will have a final volume of 10 μL (The final volume
indicated for each reaction above can be varied / increased according to need, in that case the
volume of all the reagents must be adjusted to respect the recommended final concentration).
 Take the plate to the thermocycler and start the reaction.

SYBR® Green System:

 Connect the molecular biology cabinet and leave in UV light for 10 minutes;
 Inside the cabinet, dilute the primers at a concentration of 10 μM in a microtube (stock usually
at 200 μM);
 Prepare the Mix containing the primers, adding:
 0.2 μL Pair of primers (10 μM)
 5.0 μL master mix
 2.8 μL Water RF
 Add 8 μL of reaction MIX per well to the PCR plate (96- or 384-well plate);
 Outside the cabinet, add 2 μL containing 10 ng of cDNA per reaction to a final volume of 10
μL. (The final volume of the reaction may vary as needed, so all reagent concentrations should
be adjusted.)
Application of PCR in Agriculture:
1.Plasmid Cloning:
2.Pathogen detection:

3. Grain Processing:
 PCR testing for unapproved events
 PCR testing for GM content
 PCR testing for non GM labeling
 PCR testing for presence of high-value commodity
4. Cultivar Identification of different Crops:
 Cultivars of different crops like rice markedly affect eating quality, processing suitability, and price,
identification or differentiation.
5. Identification of genetic relation of different crops.
6. PCR is equally helpful for a sensitive test for tissue typing that is crucial to organ transplantation of
Classification of organism:
 The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial
DNA data base, then the bacterium can be identified.
 Here, a rapid and reliable two-step multiplex PCR assay for identifying 14 Gram-positive anaerobic cocci (GPAC) species originally
classified in the genus Peptostreptococcus (Anaerococcus hydrogenalis )
 Bacteriumb Published primers/universal primers Al-Quds University general primers (QUGP)
 RTU8a 909 bpa 909 bp Ra 8F F1 F3 R3 F4 R4 F5 R5 F6 R6 R1b R2 R7
 Acidobacteria bacterium Y Y Y Y Y R Y Y Y Y Y
Y
 Acinetobacter sp. Y N Y Y M Y Y Y Y Y Y Y
 Aeromonas hydrophila Y N Y Y M Y Y Y Y Y Y
Y
 Agrobacterium tumefaciens N Y Y N N Y Y Y Y Y Y
Y
 Anabaena variabilisY Y Y Y Y N Y Y Y Y Y N
 Aquifex aeolicus VF5 Y N Y Y Y R Y M Y Y N
N
 Arthrobacter aurescens N Y Y N N Y Y Y Y Y Y
N
Genotypic:
 Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual
by examining the individual's DNA sequence using biological assays and comparing it to another
individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their
parents.
 Genotyping determines differences in genetic complement by comparing a DNA sequence to that of
another sample or a reference sequence .PCR can be applied for determining the genotype.Different
genotype are found in different PCR.
Mutagenesis:
 PCR mutagenesis is a method for generating site-directed mutagenesis. This method can generate
mutations (base substitutions, insertions, and deletions) from double-stranded plasmid without the need
for subcloning into M13-based bacteriophage vectors and for ssDNA rescue.
Mutation detection:
 Although many, even most, methods of mutation detection depend on polymerase chain reaction (PCR),
in the majority of techniques PCR itself does not detect the actual mutation. Rather, PCR generates an
amplicon that is then analyzed by some other method to find possible mutations within the ampli-con,
such as conformation-based techniques like single-stranded conformational polymorphism (SSCP)
analysis, denaturing gradient gel electrophoresis (DGGE), or sequencing.
Sequencing:
 Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method
involves using short DNA sequences called primers to select the portion of the genome to be amplified.
The technique can produce a billion copies of the target sequence in just a few hours.
Cancer research:
 Advances in the biological sciences and technology are providing molecular targets for diagnosing and
treating cancer. Current classifications in surgical pathology for staging malignancies are based primarily
on anatomic features (e.g., tumor-node-metastasis) and histopathology (e.g., grade).
GMO food detection:
 This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material
within the feed/food chain. The GM crop regulatory framework at the international level is evaluated
with reference to traceability and labelling. Current analytical methods for the detection, identification,
and quantification of transgenic DNA in food and feed are reviewed.
Archaeology:
 One of the problems faced by archaeologists is in identifying species from fragmented bone recovered in
sites. We felt that PCR/DNA technology offered the means by which species could be identified from
such remains. We used fragmented bones from the site of Head-Smashed-In Buffalo Jump to establish
protocols for PCR/DNA analysis.
Advantages of PCR:
 PCR in clinical diagnosis
 PCR in DNA sequencing
 PCR in Forensic medicine
 PCR in gene manipulation and expression studies
 PCR in comparative studies of genomics
 PCR in comparision with gene cloning
 Detection of genetic disorder of heridity disease
 DNA finger printing
 Analysis of ancient DNA
Advantages of PCR in Agriculture:
 Product development
 Grain processing
 Identification of fishery products
 Cultivar identification of rice
 Quantification of Fusarium culmorum in wheat and barley

Disadvantages:
 It can amplify only short fragment usually upto 10kb

 The contamination of target DNA needs to error in results

 It requires specific primer for the template to be amplified

 The cross reactivity with non-targated DNA leads to amplification of non-specific DNA

 Error rate during amplification

 Sensitivity to inhibitors