DNA Sequencing LECTURE 6: Biotechnology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)
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DNA Sequencing
LECTURE 6:
Biotechnology; 3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)National University of Sciences and Technology (NUST)
Introduction
• In 1953, two researchers, namely James Watson and Francis Crick, discovered the basic structure of DNA
• DNA is basically a long molecule that stores coded instructions for the cell
• The DNA provides a basic blueprint that is responsible for the creation and functioning of cells
Introduction
• The information contained in it dictates which cells should grow and when a particular cell should die and how cells should be structured into creating various body parts
• For example, the DNA is responsible for determining the quality of our hair, the color and the abundance, or the lack of it
DNA and Nucleotides
• DNA stands for Deoxyribonucleic acid• It is present in almost all organisms and it stores
long term information that is used to construct an organic body
• DNA comprises of a long molecule analogous to a chain, while the links of the chain are called Nucleotides
• There are four different nucleotides in the DNA, namely adenine, guanine, cytosine and thymine. They are also called as “A”, “G”, “C” and “T”
The Need for DNA Sequencing
• Forensic biology uses DNA sequences to identify the organism which it is unique to
• To isolate the genes responsible for genetic diseases like Cystic Fibrosis, Alzheimer’s disease, myotonic dystrophy, etc., which are caused by the inability of genes to work properly
• Agriculture has been helped immensely by DNA sequencing
• It has allowed scientists to make plants more resistant to insects and pests, by understanding their genes
Genome Sequencing
• Genome sequencing is determining the exact order of DNA nucleotides in the genome
• In human genome 3.08 billion nucleotides are present, 99% accurately sequenced
• DNA sequencing includes several methods and technologies
• DNA sequencing can be applied in numerous fields such as diagnostic, biotechnology and biological systems
• The first DNA sequences were obtained in the early 1970s
Avery: Proposes DNA as ‘Genetic Material’
Watson & Crick: Double Helix Structure of DNA
Holley: Sequences Yeast tRNAAla
1870
1953
1940
1965
1970
1977
1980
1990
2002
Miescher: Discovers DNA
Wu: Sequences Cohesive End DNA
Sanger: Dideoxy Chain TerminationGilbert: Chemical Degradation
Messing: M13 Cloning
Hood et al.: Partial Automation
• Cycle Sequencing • Improved Sequencing Enzymes• Improved Fluorescent Detection Schemes
1986
• Next Generation Sequencing•Improved enzymes and chemistry•Improved image processing
Date Cost per Mb of DNA Sequence Cost per GenomeSeptember-2001 $5,292.39 $95,263,072March-2002 $3,898.64 $70,175,437September-2002 $3,413.80 $61,448,422March-2003 $2,986.20 $53,751,684October-2003 $2,230.98 $40,157,554January-2004 $1,598.91 $28,780,376April-2004 $1,135.70 $20,442,576July-2004 $1,107.46 $19,934,346October-2004 $1,028.85 $18,519,312January-2005 $974.16 $17,534,970April-2005 $897.76 $16,159,699July-2005 $898.90 $16,180,224October-2005 $766.73 $13,801,124January-2006 $699.20 $12,585,659April-2006 $651.81 $11,732,535July-2006 $636.41 $11,455,315October-2006 $581.92 $10,474,556
Cost of DNA Sequencing
January-2007 $522.71 $9,408,739
April-2007 $502.61 $9,047,003
July-2007 $495.96 $8,927,342
October-2007 $397.09 $7,147,571
January-2008 $102.13 $3,063,820
April-2008 $15.03 $1,352,982
July-2008 $8.36 $752,080
October-2008 $3.81 $342,502
January-2009 $2.59 $232,735
April-2009 $1.72 $154,714
July-2009 $1.20 $108,065
October-2009 $0.78 $70,333
January-2010 $0.52 $46,774
April-2010 $0.35 $31,512
July-2010 $0.35 $31,125
October-2010 $0.32 $29,092
January-2011 $0.23 $20,963
April-2011 $0.19 $16,712
July-2011 $0.12 $10,497
Jan-2012 (EST) $0.09 $7,950
Cost of DNA Sequencing
Sequencing Methods
• To determine the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used1. Maxam and Gilbert; Chemical Sequencing2. Sanger; Chain Termination Sequencing
• These two are conventional methods• Robotics and automated sequencing are
based on these methods
1- Maxam and Gilbert Method
• In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases
I. Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP)
II. Purification of the DNA fragment to be sequencedIII. Chemical treatment generates breaks in DNAIV. Run on the gel
Chemical Modification and Cleavage
• Ploy nucleotide Kinase radioactive label at one 5' end of the DNA using gamma-32P
5 ′ G A C G T G C A A C G A A 3′
32P 5 ′ G A C G T G C A A C G A A 3′
• Dimethyl sulphate• Purine– Adenine– Guanine
• Only DMS------- G• DMS+ Formic acid-------G+A• Piperidine
Chemical Modification and Cleavage
DMS
G
GG
G
FA
GA
GG
AG
AA
H
CTT
C
TC
CT
H+S
CC
CC
Maxam Gilbert Sequencing
32P 5 ′ G A C G T G C A A C G A 3′
• Hydrazine• Pyrimidine– Cytocine– Thymidine
• Hydrazine----- C+T• Hydrazine + NaCl--------C
Chemical Modification and Cleavage
DMS
G
GG
G
FA
GA
GG
AG
AA
H
CTT
C
TC
CT
H+S
CC
CC
Maxam Gilbert Sequencing
32P 5 ′ G A C G T G C A A C G A 3′
Sequencing gels are read from bottom to top (5 to 3 ).′ ′
G G+A T+C C
3′AGCAACGTGCAG5′
Longer fragments
Shortest fragmentsG
A
Maxam-Gilbert Sequencing
32P 5 ′ G A C G T G C A A C G A 3′
2- Sanger; Chain Termination Sequencing
• Its PCR based method• A modified DNA replication reaction• Growing chains are terminated by
dideoxynucleotides
The 3 -OH group necessary for formation of the phosphodiester bond is missing in ddNTPs′
ddATP + ddAfour dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddCfour dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC
ddGTP + dAddGfour dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddTfour dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
Sanger; Chain Termination Sequencing
A G C T G C C C G
Sequencing gels are read from bottom to top (5 to 3 )′ ′
G A T C
3′GGTAAATCATG5′
Longer fragments
Shorter fragmentsddG
ddG
Chain Termination Sequencing
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