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