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DNA Sequencing
Institute of Lifelong Learning, University of Delhi 1
Lesson Prepared Under MHRD project “ National Mission on Education Through
ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: DNA Sequencing
Lesson Developer: Vinee Khanna
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Reviewer: Dr. Rahul Kumar
Repository of Tomato Genomic Resources
Department of Plant Sciences, School of Life Sciences
University of Hyderabad
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Page 2
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 1
Lesson Prepared Under MHRD project “ National Mission on Education Through
ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: DNA Sequencing
Lesson Developer: Vinee Khanna
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Reviewer: Dr. Rahul Kumar
Repository of Tomato Genomic Resources
Department of Plant Sciences, School of Life Sciences
University of Hyderabad
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 2
Chapter: DNA Sequencing
Table of Contents
Chapter: DNA Sequencing
? Introduction
? Methods of sequencing
? Sanger’s method
? Shotgun Approach
? Advantages
? Disadvantages
? Primer walking
? Advantages
? Disadvantages
? Maxam and Gilbert’s method of sequencing
? Pyrosequencing
? Advantages
? Disadvantages
? Applications
? Cycle sequencing
? Next Generation Sequencing
? Sequencers in NGS
? Roche/454 FLX pyrosequencer
? Ilumina Genome Analyzer
? Applied Biosystems SOLiDTM
Sequencer
Page 3
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 1
Lesson Prepared Under MHRD project “ National Mission on Education Through
ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: DNA Sequencing
Lesson Developer: Vinee Khanna
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Reviewer: Dr. Rahul Kumar
Repository of Tomato Genomic Resources
Department of Plant Sciences, School of Life Sciences
University of Hyderabad
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 2
Chapter: DNA Sequencing
Table of Contents
Chapter: DNA Sequencing
? Introduction
? Methods of sequencing
? Sanger’s method
? Shotgun Approach
? Advantages
? Disadvantages
? Primer walking
? Advantages
? Disadvantages
? Maxam and Gilbert’s method of sequencing
? Pyrosequencing
? Advantages
? Disadvantages
? Applications
? Cycle sequencing
? Next Generation Sequencing
? Sequencers in NGS
? Roche/454 FLX pyrosequencer
? Ilumina Genome Analyzer
? Applied Biosystems SOLiDTM
Sequencer
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 3
? Advantages
? Applications of NGS
? Summary
? Exercise/Practice
? Glossary
? References
? Further reading
Page 4
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 1
Lesson Prepared Under MHRD project “ National Mission on Education Through
ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: DNA Sequencing
Lesson Developer: Vinee Khanna
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Reviewer: Dr. Rahul Kumar
Repository of Tomato Genomic Resources
Department of Plant Sciences, School of Life Sciences
University of Hyderabad
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 2
Chapter: DNA Sequencing
Table of Contents
Chapter: DNA Sequencing
? Introduction
? Methods of sequencing
? Sanger’s method
? Shotgun Approach
? Advantages
? Disadvantages
? Primer walking
? Advantages
? Disadvantages
? Maxam and Gilbert’s method of sequencing
? Pyrosequencing
? Advantages
? Disadvantages
? Applications
? Cycle sequencing
? Next Generation Sequencing
? Sequencers in NGS
? Roche/454 FLX pyrosequencer
? Ilumina Genome Analyzer
? Applied Biosystems SOLiDTM
Sequencer
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 3
? Advantages
? Applications of NGS
? Summary
? Exercise/Practice
? Glossary
? References
? Further reading
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 4
Learning outcomes
After reading this chapter, the reader should be able to understand the following:
? Genome sequencing and its basics.
? The principle behind DNA sequencing.
? Various methods of sequencing, including Next generation sequencing (NGS).
? Applications of sequencing.
Introduction
DNA sequencing tells us about the precise sequence of nucleotides in the sample of DNA.
The oldest method of sequencing is Sanger’s method, which was first introduced in the year
1977. There have been many modifications in this method since then and many
technologies have been introduced to improve the quality, time, length and cost of reads.
The latest technology introgression in this field has been that of Next Generation
Sequencing (NGS), which is a rapid, high throughput method. We shall be discussing
various methods of sequencing in detail.
What is a genome?
It is a list of instructions which encodes the formation of DNA which in turn constitutes
an organism. For example, the human genome is made of 3 billion bases of DNA which
are arranged into 24 chromosomes.
Page 5
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 1
Lesson Prepared Under MHRD project “ National Mission on Education Through
ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: DNA Sequencing
Lesson Developer: Vinee Khanna
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Reviewer: Dr. Rahul Kumar
Repository of Tomato Genomic Resources
Department of Plant Sciences, School of Life Sciences
University of Hyderabad
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi, South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 2
Chapter: DNA Sequencing
Table of Contents
Chapter: DNA Sequencing
? Introduction
? Methods of sequencing
? Sanger’s method
? Shotgun Approach
? Advantages
? Disadvantages
? Primer walking
? Advantages
? Disadvantages
? Maxam and Gilbert’s method of sequencing
? Pyrosequencing
? Advantages
? Disadvantages
? Applications
? Cycle sequencing
? Next Generation Sequencing
? Sequencers in NGS
? Roche/454 FLX pyrosequencer
? Ilumina Genome Analyzer
? Applied Biosystems SOLiDTM
Sequencer
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 3
? Advantages
? Applications of NGS
? Summary
? Exercise/Practice
? Glossary
? References
? Further reading
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 4
Learning outcomes
After reading this chapter, the reader should be able to understand the following:
? Genome sequencing and its basics.
? The principle behind DNA sequencing.
? Various methods of sequencing, including Next generation sequencing (NGS).
? Applications of sequencing.
Introduction
DNA sequencing tells us about the precise sequence of nucleotides in the sample of DNA.
The oldest method of sequencing is Sanger’s method, which was first introduced in the year
1977. There have been many modifications in this method since then and many
technologies have been introduced to improve the quality, time, length and cost of reads.
The latest technology introgression in this field has been that of Next Generation
Sequencing (NGS), which is a rapid, high throughput method. We shall be discussing
various methods of sequencing in detail.
What is a genome?
It is a list of instructions which encodes the formation of DNA which in turn constitutes
an organism. For example, the human genome is made of 3 billion bases of DNA which
are arranged into 24 chromosomes.
DNA Sequencing
Institute of Lifelong Learning, University of Delhi 5
Video:http://www.dnalc.org/view/16812-Animation-39-A-genome-is-an-entire-set-of-
genes-.html (cc)
The DNA molecule is made of a sugar backbone and 3’-5’ OH (hydroxyl) residues. The sugar
backbone is made up by nucleotides. These nucleotides consist of a five carbon sugar
deoxyribose, a nitrogenous base and a phosphate group. The nitrogenous bases may be
Adenine, Cytosine, Guanine or Thymine. The 3’-5’ OH group is called the deoxy- group and
is essential for the chain elongation. In DNA sequencing these bases are read in a DNA
fragment.
Figure: Illustration of structure of a DNA molecule and its constituent bases.
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FAQs on Lecture 17 - DNA Sequencing - Plant Biotechnology - Botany
| 1. What is DNA sequencing? |  |
Ans. DNA sequencing is a process that determines the precise order of nucleotides in a DNA molecule. It allows scientists to analyze and understand the genetic information encoded in the DNA sequence.
| 2. How does DNA sequencing work? | |
Ans. DNA sequencing involves several steps. First, the DNA sample is prepared, which may include amplification and purification. Then, a DNA sequencing machine reads the sequence of nucleotides by detecting the incorporation of fluorescently labeled nucleotides. The machine generates a series of signals that are converted into a DNA sequence using specialized software.
| 3. What are the applications of DNA sequencing in botany? | |
Ans. DNA sequencing has various applications in botany. It can be used to identify plant species, analyze genetic diversity, study plant evolution, and understand plant responses to environmental factors. It also helps in developing new crop varieties, identifying genes responsible for specific traits, and studying plant-pathogen interactions.
| 4. What are the benefits of using DNA sequencing in botany research? | |
Ans. DNA sequencing in botany research provides several benefits. It allows for accurate species identification, even in cases where plants have similar morphological characteristics. It helps in understanding the relationships between different plant species and their evolutionary history. It also enables the discovery of new genetic markers and genes associated with desirable traits, facilitating crop improvement and conservation efforts.
| 5. What are the limitations of DNA sequencing in botany? | |
Ans. Although DNA sequencing is a powerful tool in botany, it does have limitations. One limitation is the cost and time required for sequencing large genomes. Another limitation is the difficulty in sequencing repetitive regions of the genome accurately. Additionally, DNA sequencing may not capture the full complexity of gene regulation and interactions within a plant. Therefore, it is often combined with other techniques to obtain a comprehensive understanding of plant biology.
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