Page 1
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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