Lecture 16 - Polymerase Chain Reaction (PCR) | Plant Biotechnology - Botany PDF Download

 Page 1


Polymerase Chain Reaction (PCR) 
Institute of lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Polymerase Chain Reaction (PCR) 
Lesson Developer: Dr. Prabhavathi 
Department/College: Department of Botany, Shivaji College, 
University of Delhi 
 
Page 2


Polymerase Chain Reaction (PCR) 
Institute of lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Polymerase Chain Reaction (PCR) 
Lesson Developer: Dr. Prabhavathi 
Department/College: Department of Botany, Shivaji College, 
University of Delhi 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
1 
Table of Contents 
Chapter: Polymerase Chain Reaction 
? Introduction 
? Components of PCR 
? Template 
? Primers 
? Properties of primers 
? DNA polymerase 
? dNTPs (deoxynucleotide triphosphates) 
? Buffer 
? Divalent cations 
? Thermal cycler 
? Procedure 
? Denaturation 
? Annealing 
? Extension 
? Variants of PCR 
? Long accurate- PCR (LA-PCR)  
? Inverse PCR 
? Hot Start PCR 
? Nested PCR 
? Real time-q PCR 
? RT- PCR (Reverse Transcriptase PCR) 
? Multiplex PCR 
? Applications 
? Diagnosis of Genetic diseases 
? Detection of the bacterial and viral infections 
? Forensic studies 
? Research laboratories 
? Gene cloning  
? Identification of transgenics 
? Gene expression studies  
? DNA Sequencing 
? Marker-assisted plant breeding 
Page 3


Polymerase Chain Reaction (PCR) 
Institute of lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Polymerase Chain Reaction (PCR) 
Lesson Developer: Dr. Prabhavathi 
Department/College: Department of Botany, Shivaji College, 
University of Delhi 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
1 
Table of Contents 
Chapter: Polymerase Chain Reaction 
? Introduction 
? Components of PCR 
? Template 
? Primers 
? Properties of primers 
? DNA polymerase 
? dNTPs (deoxynucleotide triphosphates) 
? Buffer 
? Divalent cations 
? Thermal cycler 
? Procedure 
? Denaturation 
? Annealing 
? Extension 
? Variants of PCR 
? Long accurate- PCR (LA-PCR)  
? Inverse PCR 
? Hot Start PCR 
? Nested PCR 
? Real time-q PCR 
? RT- PCR (Reverse Transcriptase PCR) 
? Multiplex PCR 
? Applications 
? Diagnosis of Genetic diseases 
? Detection of the bacterial and viral infections 
? Forensic studies 
? Research laboratories 
? Gene cloning  
? Identification of transgenics 
? Gene expression studies  
? DNA Sequencing 
? Marker-assisted plant breeding 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
2 
? Summary 
? Exercises 
? Glossary 
? References 
 
 
 
    
    
    
     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 4


Polymerase Chain Reaction (PCR) 
Institute of lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Polymerase Chain Reaction (PCR) 
Lesson Developer: Dr. Prabhavathi 
Department/College: Department of Botany, Shivaji College, 
University of Delhi 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
1 
Table of Contents 
Chapter: Polymerase Chain Reaction 
? Introduction 
? Components of PCR 
? Template 
? Primers 
? Properties of primers 
? DNA polymerase 
? dNTPs (deoxynucleotide triphosphates) 
? Buffer 
? Divalent cations 
? Thermal cycler 
? Procedure 
? Denaturation 
? Annealing 
? Extension 
? Variants of PCR 
? Long accurate- PCR (LA-PCR)  
? Inverse PCR 
? Hot Start PCR 
? Nested PCR 
? Real time-q PCR 
? RT- PCR (Reverse Transcriptase PCR) 
? Multiplex PCR 
? Applications 
? Diagnosis of Genetic diseases 
? Detection of the bacterial and viral infections 
? Forensic studies 
? Research laboratories 
? Gene cloning  
? Identification of transgenics 
? Gene expression studies  
? DNA Sequencing 
? Marker-assisted plant breeding 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
2 
? Summary 
? Exercises 
? Glossary 
? References 
 
 
 
    
    
    
     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
3 
Introduction 
Polymerase Chain Reaction (PCR) is one of the principal techniques used in molecular 
biology. It is a simple, rapid and inexpensive tool used to a generate millions of copies of 
DNA from fairly small amount of DNA. This technique was developed in 1983 by Kary B. 
Mullis. In recognition of their contribution to science, Mullis along with Michael Smith was 
awarded Nobel Prize (1993) in Chemistry. 
 
 
 
 
 
 
 
 
 
 
Figure: A. Kary Mullis; B Michael Smith 
Source:http://en.wikipedia.org/wiki/Kary_Mullis, 
http://en.wikipedia.org/wiki/Michael_Smith_%28chemist%29 (CC) 
The technique has wide range of applications in biological research, like gene cloning, 
sequencing, forensic sciences and diagnosis of different diseases. 
 
 
 
Video:https://www.youtube.com/watch?v=2KoLnIwoZKU&list=PL854F3A5D92AFC121 (CC) 
A 
B 
Page 5


Polymerase Chain Reaction (PCR) 
Institute of lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
 
 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Polymerase Chain Reaction (PCR) 
Lesson Developer: Dr. Prabhavathi 
Department/College: Department of Botany, Shivaji College, 
University of Delhi 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
1 
Table of Contents 
Chapter: Polymerase Chain Reaction 
? Introduction 
? Components of PCR 
? Template 
? Primers 
? Properties of primers 
? DNA polymerase 
? dNTPs (deoxynucleotide triphosphates) 
? Buffer 
? Divalent cations 
? Thermal cycler 
? Procedure 
? Denaturation 
? Annealing 
? Extension 
? Variants of PCR 
? Long accurate- PCR (LA-PCR)  
? Inverse PCR 
? Hot Start PCR 
? Nested PCR 
? Real time-q PCR 
? RT- PCR (Reverse Transcriptase PCR) 
? Multiplex PCR 
? Applications 
? Diagnosis of Genetic diseases 
? Detection of the bacterial and viral infections 
? Forensic studies 
? Research laboratories 
? Gene cloning  
? Identification of transgenics 
? Gene expression studies  
? DNA Sequencing 
? Marker-assisted plant breeding 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
2 
? Summary 
? Exercises 
? Glossary 
? References 
 
 
 
    
    
    
     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
3 
Introduction 
Polymerase Chain Reaction (PCR) is one of the principal techniques used in molecular 
biology. It is a simple, rapid and inexpensive tool used to a generate millions of copies of 
DNA from fairly small amount of DNA. This technique was developed in 1983 by Kary B. 
Mullis. In recognition of their contribution to science, Mullis along with Michael Smith was 
awarded Nobel Prize (1993) in Chemistry. 
 
 
 
 
 
 
 
 
 
 
Figure: A. Kary Mullis; B Michael Smith 
Source:http://en.wikipedia.org/wiki/Kary_Mullis, 
http://en.wikipedia.org/wiki/Michael_Smith_%28chemist%29 (CC) 
The technique has wide range of applications in biological research, like gene cloning, 
sequencing, forensic sciences and diagnosis of different diseases. 
 
 
 
Video:https://www.youtube.com/watch?v=2KoLnIwoZKU&list=PL854F3A5D92AFC121 (CC) 
A 
B 
Polymerase Chain Reaction 
Institute of Lifelong Learning, University of Delhi  
 
4 
 
Discovery of PCR 
In 1983, Mullis was working for one of the biotechnology companies, Cetus Corporation as 
a chemist. One day he had an idea that a pair of primers can be used to copy the DNA 
sequence using DNA polymerase. Mullis succeeded in demonstrating PCR in December 16, 
1983. Other Cetus scientists, Randall Saiki and Henry Erlich, also worked on the PCR 
project to test whether PCR could amplify a specific human gene (betaglobin) from 
genomic DNA. Saiki and Erlich published first paper on utilization of the technique. At the 
same time, Mullis was still working on a paper that would describe PCR itself. The 
polymerase that was used for the amplification destroyed at high temperature. In 1986, 
Saiki started to use Thermophilus aquaticus (Taq) DNA polymerase to amplify segments 
of DNA. The Taq polymerase was heat resistant and would only need to be added once, 
thus making the technique dramatically more affordable and subject to automation. This 
has created revolution in biochemistry,molecular biology, genetics, medicine and 
forensics. 
Source: https://en.wikipedia.org/wiki/Kary_Mullis