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16 views11 pages

PCR_cutm

Uploaded by

victor.pradhan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PCR

THERMAL CYCLER

VICTOR PRADHAN
INTRODUCTION

• The polymerase chain reaction (PCR) was originally developed in 1983 by


the American biochemist Kary Mullis
• PCR is used in molecular biology to make many copies of (amplify) small
sections of DNA .
• Using PCR it is possible to generate thousands to millions of copies of a
particular section of DNA from a very small amount of DNA.
• Polymerases are enzymes that catalyze the synthesis of DNA or RNA
polymers whose sequence is complementary to the original template.

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TYPES OF PCR

• Real-time PCR
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR
• Nested PCR
• Long-range PCR
• Single-cell PCR
• Fast-cycling PCR
• Methylation-specific PCR (MSP)
• Hot start PCR

[Image source1- (THERMAL CYCLER)


https://en.wikipedia.org/wiki/File:Biometra_Trio_Touchscreen_.jpg]

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OVERVIEW

[Image source 2 (PICTORIAL REPRESENTATION)- https://images.app.goo.gl/bevjDxr3jY7HaMZaA]


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STEPS
• DENATURATION
• During this stage the cocktail containing the template DNA and all the other core
ingredients is heated to 94-95 ⁰C.
• The high temperature causes the hydrogen bonds between the bases in two
strands of template DNA to break and the two strands to separate.
• This results in two single strands of DNA, which will act as templates for the
production of the new strands of DNA.
• It is important that the temperature is maintained at this stage for long enough to
ensure that the DNA strands have separated completely.
• This usually takes between 15-30 seconds.

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• ANNEALING
• During this stage the reaction is cooled to 50-65 ⁰C. This enables the primers to
attach to a specific location on the single-stranded template DNA by way of
hydrogen bonding.
• Primers are single strands of DNA.
• The primers are designed to be complementary in sequence to short sections of
DNA on each end of the sequence to be copied.
• The two separated strands of DNA are complementary and run in opposite
directions (from one end - the 5’ end – to the other - the 3’ end); as a result, there
are two primers – a forward primer and a reverse primer.
• This step usually takes about 10-30 seconds.

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• EXTENSION
• During this final step, the heat is increased to 72 ⁰C to enable the new DNA to be
made by a special DNA polymerase enzyme which adds DNA bases.
• The bacteria's DNA polymerase is very stable at high temperatures, which means it
can withstand the temperatures needed to break the strands of DNA apart in the
denaturing stage of PCR.
• DNA polymerase from most other organisms would not be able to withstand these
high temperatures, for example, human polymerase works ideally at 37˚C (body
temperature).
• 72⁰C is the optimum temperature for the Taq polymerase to build the
complementary strand. It attaches to the primer and then adds DNA bases to the
single strand one-by-one in the 5’ to 3’ direction.

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INSTRUMENTATION
• BUFFER-
• Tris and KCL(>50mM can inhibit Taq Pol)
buffer can be used, helps in facilitating the
primer binding and annealing
• MgCl2-
• Also required for primer binding. High
amount leads to non specific binding and little
amount leads to low yield.
• dNTP-
• stands for deoxyribose nucleotide
triphosphate employed in PCR to expand
the growing DNA strand. The function of
dNTPs in PCR is to expand the growing DNA
strand with the help of Taq DNA polymerase.

[Image source 3 (MASTER MIX) - https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/pcr-mast

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APPLICATIONS OF PCR IN AGRICULTURE

•Crop Improvement
•Marker-assisted breeding for desirable traits. •Food Safety
•Genetic mapping to develop improved crop varieties. •Detecting contaminants like mycotoxins in crops.
•Disease Diagnostics •Verifying authenticity and origin of agricultural products.
•Early detection of plant pathogens. •Biodiversity Conservation
•Quarantine and biosecurity for invasive species. •Studying genetic diversity in crops and wild relatives.
•GMO Detection •Identifying endangered plant species.
•Identification and quantification of GMOs. •Climate-Resilient Agriculture
•Compliance with regulatory standards. •Identifying genes for stress tolerance (e.g., drought, salinity)
•Soil and Microbial Analysis •Studying epigenetic responses to environmental changes.
•Identifying beneficial microbes for soil health. •Biotechnology and Precision Agriculture
•Monitoring microbial diversity for bioremediation. •Validation of gene editing (e.g., CRISPR-Cas9).
•Pest Management •Soil health and crop condition monitoring.
•Early detection of insect pests.
•Monitoring pesticide resistance in pests.

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THANK
YOU

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