SlideShare a Scribd company logo
1 of 21
DNA SEQUENCING
SANGER’S METHOD
HARSHA JOSEPH
MSc LIFE SCIENCE
What is sequencing?
DNA sequencing is the process of determining
the sequence of nucleotide bases (As, Ts, Cs,
and Gs) in a piece of DNA. Today, with the
right equipment and materials, sequencing a
short piece of DNA is relatively
straightforward.
Sequencing an entire genome (all of an
organism’s DNA) remains a complex task. It
requires breaking the DNA of the genome into
many smaller pieces, sequencing the pieces,
and assembling the sequences into a single
long "consensus." However, thanks to new
methods that have been developed over the
past two decades, genome sequencing is now
much faster and less expensive than it was
during the Human Genome Project.
Sanger sequencing: The chain
termination method
INTRODUCTION
• The DNA sequencing method developed by Fred Sanger
forms the basis of automated "cycle" sequencing reactions
today.
• In the 1980s, two key developments allowed researchers to
believe that sequencing the entire genome could be
possible.
• The first was a technique called polymerase chain reaction
(PCR) that enabled many copies of DNA sequence to be
quickly and accurately produced.
• The second, an automated method of DNA sequencing,
built upon the chemistry of PCR and the sequencing
process developed by Frederick Sanger in 1977.
• Sanger sequencing method of DNA
sequencing was first commercialized by
Applied Biosystems.
• It was the most widely used sequencing
method for approximately 40 years.
• Regions of DNA up to about 900 base pairs in
length are routinely sequenced using this
method.
• In the Human Genome Project, Sanger
sequencing was used to determine the sequences
of many relatively small fragments of human
DNA.
• The fragments were aligned based on
overlapping portions to assemble the sequences
of larger regions of DNA and, eventually, entire
chromosomes.
Requirements for Sanger sequencing
Fred Sanger’s method of DNA sequencing was based on Arthur Kornberg’s
earlier work on DNA Replication. A new DNA strand is synthesized using an
existing strand as a template.Sanger sequencing involves making many
copies of a target DNA region. Its ingredients are similar to those needed
for DNA replication in an organism, or for polymerase chain reaction. They
include:
• A DNA polymerase enzyme
• A primer, which is a short piece of single-stranded DNA that binds
to the template DNA and acts as a "starter" for the polymerase
• The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• The template DNA to be sequenced
However, a Sanger sequencing reaction also contains a unique
ingredient:
• Dideoxy, or chain-terminating, versions of all four nucleotides
(ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of
dye.
Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one
key difference: they lack a hydroxyl group on the 3’ carbon of the sugar ring. In
a regular nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a new
nucleotide to be added to an existing chain.
PRINCIPLE
• The 5’ carbon of an “incoming” deoxynucleotide
(dNTP) is joined to the 3’ carbon at the end of the
chain. Hydroxyl groups in each position form
ester linkages with a central phosphate. In this
way, the nucleotide chain elongates.
• The key to Sanger’s sequencing method is the
peculiar chemistry of dideoxynucleotides
(ddNTP). Like a deoxynucleotide, a ddNTP is
incorporated into a chain by forming a
phosphodiester linkage at its 5’ end.
• However, the ddNTP lacks a 3’ hydroxyl group
(OH) necessary to form the linkage with an
incoming nucleotide. So the addition of a ddNTP
halts elongation.
Method
• To sequence DNA, four separate reactions are
necessary – one to provide sequence
information about each of the nucleotides.
Each reaction contains: template DNA, a short
primer (about 20 nucleotides), DNA
polymerase, and the four dNTPs (one
radioactively labeled).
• One type of ddNTP – A, T, C, or G – is added
to each.
The mixture is first heated to denature the
template DNA (separate the strands), then
cooled so that the primer can bind to the single-
stranded template.
The DNA Polymerase makes no distinction between
dNTPs or ddNTPs. Each time a ddNTP is incorporated, in
this case ddATP, synthesis is “terminated” and a DNA
strand of a discrete size is generated. In this sequencing
example, the ddATP (purple) has terminated the
reaction. The dATP happens to be the radioactive
tracer, but this has no effect on elongation.
Because billions of DNA molecules are present,
the elongation reaction can be terminated at
any adenine position. This results in collections
of DNA strands of different lengths. The same is
true for the other three terminator reactions.
• Each reaction is then loaded into a separate lane
of a polyacrylamide gel containing urea, which
prevents the DNA strands from renaturing during
electrophoesis.
• Ionized phosphates give the DNA molecule a
negative charge, so DNA migrate toward the
positive pole of an electric field. The movement
of DNA molecules through the polyacrylamide
matrix is size dependent.
• Over the course of electrophoresis, shorter DNA
molecules will move further down the gel than
larger ones. Millions of terminated molecules of
the same size will migrate to the same place and
“band” in the gel.
After electrophoresis, the gel is sandwiched against X-ray film. The
radioactive adenine in the synthesized DNA emit beta particles that
expose the film, making a record of the positions of DNA bands in the
gel. The sequencing gel is then read from bottom to top. The sequence
of bands in the various terminator lanes gives the sequence of
nucleotides in the template DNA.
Another method for reading the
sequence:
• The process of replication is repeated in a
number of cycles. By the time the cycling is
complete, it’s virtually guaranteed that a dideoxy
nucleotide will have been incorporated at every
single position of the target DNA in at least one
reaction. That is, the tube will contain fragments
of different lengths, ending at each of the
nucleotide positions in the original DNA. The
ends of the fragments will be labeled with dyes
that indicate their final nucleotide.
• After the reaction is done, the fragments are run
through a long, thin tube containing a gel matrix in a
process called capillary gel electrophoresis.
• Short fragments move quickly through the pores of the
gel, while long fragments move more slowly. As each
fragment crosses the “finish line” at the end of the
tube, it’s illuminated by a laser, allowing the attached
dye to be detected.
• From the colors of dyes registered one after another on
the detector, the sequence of the original piece of DNA
can be built up one nucleotide at a time. The data
recorded by the detector consist of a series of peaks in
fluorescence intensity. The DNA sequence is read from
the peaks in the chromatogram.
Uses and limitations
• Sanger sequencing gives high-quality sequence for
relatively long stretches of DNA (up to about 900 base
pairs). It's typically used to sequence individual pieces
of DNA, such as bacterial plasmids or DNA copied in
PCR.
• However, Sanger sequencing is expensive and
inefficient for larger-scale projects, such as the
sequencing of an entire genome or metagenome (the
“collective genome” of a microbial community). For
tasks such as these, new, large-scale sequencing
techniques are faster and less expensive.
THANK YOU !

More Related Content

What's hot

What's hot (20)

Genome mapping
Genome mapping Genome mapping
Genome mapping
 
Artificial chromosomes - YAC and BAC
Artificial chromosomes - YAC and BACArtificial chromosomes - YAC and BAC
Artificial chromosomes - YAC and BAC
 
DNA footprinting
DNA footprintingDNA footprinting
DNA footprinting
 
Genome sequencing
Genome sequencingGenome sequencing
Genome sequencing
 
Genomic library
Genomic libraryGenomic library
Genomic library
 
Linker, Adaptor, Homopolymeric Tailing & Terminal Transferase
Linker, Adaptor, Homopolymeric Tailing & Terminal TransferaseLinker, Adaptor, Homopolymeric Tailing & Terminal Transferase
Linker, Adaptor, Homopolymeric Tailing & Terminal Transferase
 
SAGE (Serial analysis of Gene Expression)
SAGE (Serial analysis of Gene Expression)SAGE (Serial analysis of Gene Expression)
SAGE (Serial analysis of Gene Expression)
 
Molecular probes
Molecular probesMolecular probes
Molecular probes
 
Dna sequencing and its types
Dna sequencing and its typesDna sequencing and its types
Dna sequencing and its types
 
Northern, southern and western blotting
Northern, southern and western blottingNorthern, southern and western blotting
Northern, southern and western blotting
 
Phagemid vector
Phagemid vectorPhagemid vector
Phagemid vector
 
Gene silencing
Gene silencing Gene silencing
Gene silencing
 
RAPD, RFLP
RAPD, RFLPRAPD, RFLP
RAPD, RFLP
 
MODIFYING ENZYMES
MODIFYING ENZYMESMODIFYING ENZYMES
MODIFYING ENZYMES
 
Mitochondrial DNA
Mitochondrial DNAMitochondrial DNA
Mitochondrial DNA
 
Gene sequencing methods
Gene sequencing methodsGene sequencing methods
Gene sequencing methods
 
Random Amplified polymorphic DNA. RAPD
Random Amplified polymorphic DNA. RAPDRandom Amplified polymorphic DNA. RAPD
Random Amplified polymorphic DNA. RAPD
 
DNA microarray
DNA microarrayDNA microarray
DNA microarray
 
DNA Sequencing
DNA SequencingDNA Sequencing
DNA Sequencing
 
Rflp presentation
Rflp presentationRflp presentation
Rflp presentation
 

Similar to DNA Sequencing- Sanger's Method

Similar to DNA Sequencing- Sanger's Method (20)

Sanger sequencing
Sanger sequencingSanger sequencing
Sanger sequencing
 
Genome sequencing
Genome sequencing Genome sequencing
Genome sequencing
 
Sanger-Shortgun sequencing.pdf
Sanger-Shortgun sequencing.pdfSanger-Shortgun sequencing.pdf
Sanger-Shortgun sequencing.pdf
 
Dna sequencing techniques
Dna sequencing techniquesDna sequencing techniques
Dna sequencing techniques
 
Dna sequencing by Dideoxy method
Dna sequencing by Dideoxy methodDna sequencing by Dideoxy method
Dna sequencing by Dideoxy method
 
Sequencing
SequencingSequencing
Sequencing
 
dna sequencing methods
 dna sequencing methods dna sequencing methods
dna sequencing methods
 
Dna sequencing methods
Dna sequencing methodsDna sequencing methods
Dna sequencing methods
 
DNA sequencing
DNA sequencingDNA sequencing
DNA sequencing
 
Gene Sequencing
Gene SequencingGene Sequencing
Gene Sequencing
 
DNA sequencing
DNA sequencingDNA sequencing
DNA sequencing
 
DND sequencing
DND sequencingDND sequencing
DND sequencing
 
Next generation sequencing
Next generation sequencingNext generation sequencing
Next generation sequencing
 
Sanger sequencing
Sanger sequencingSanger sequencing
Sanger sequencing
 
Sanger’s Method of Sequencing (1).pptx
Sanger’s Method of Sequencing (1).pptxSanger’s Method of Sequencing (1).pptx
Sanger’s Method of Sequencing (1).pptx
 
DNA SEQUENCE ANALYSIS.pdf
DNA SEQUENCE ANALYSIS.pdfDNA SEQUENCE ANALYSIS.pdf
DNA SEQUENCE ANALYSIS.pdf
 
Dna synthesis & sequencing
Dna synthesis & sequencingDna synthesis & sequencing
Dna synthesis & sequencing
 
DNA SEQUENCING (1).pptx
DNA SEQUENCING (1).pptxDNA SEQUENCING (1).pptx
DNA SEQUENCING (1).pptx
 
GENE ISOLATION AND SEQUENCING.pdf
GENE ISOLATION AND SEQUENCING.pdfGENE ISOLATION AND SEQUENCING.pdf
GENE ISOLATION AND SEQUENCING.pdf
 
DNA sequencing.pptx
DNA sequencing.pptxDNA sequencing.pptx
DNA sequencing.pptx
 

Recently uploaded

Human genetics..........................pptx
Human genetics..........................pptxHuman genetics..........................pptx
Human genetics..........................pptxSilpa
 
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....muralinath2
 
Phenolics: types, biosynthesis and functions.
Phenolics: types, biosynthesis and functions.Phenolics: types, biosynthesis and functions.
Phenolics: types, biosynthesis and functions.Silpa
 
300003-World Science Day For Peace And Development.pptx
300003-World Science Day For Peace And Development.pptx300003-World Science Day For Peace And Development.pptx
300003-World Science Day For Peace And Development.pptxryanrooker
 
Factory Acceptance Test( FAT).pptx .
Factory Acceptance Test( FAT).pptx       .Factory Acceptance Test( FAT).pptx       .
Factory Acceptance Test( FAT).pptx .Poonam Aher Patil
 
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune Waterworlds
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune WaterworldsBiogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune Waterworlds
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune WaterworldsSérgio Sacani
 
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry Areesha Ahmad
 
biology HL practice questions IB BIOLOGY
biology HL practice questions IB BIOLOGYbiology HL practice questions IB BIOLOGY
biology HL practice questions IB BIOLOGY1301aanya
 
Cyanide resistant respiration pathway.pptx
Cyanide resistant respiration pathway.pptxCyanide resistant respiration pathway.pptx
Cyanide resistant respiration pathway.pptxSilpa
 
LUNULARIA -features, morphology, anatomy ,reproduction etc.
LUNULARIA -features, morphology, anatomy ,reproduction etc.LUNULARIA -features, morphology, anatomy ,reproduction etc.
LUNULARIA -features, morphology, anatomy ,reproduction etc.Silpa
 
Module for Grade 9 for Asynchronous/Distance learning
Module for Grade 9 for Asynchronous/Distance learningModule for Grade 9 for Asynchronous/Distance learning
Module for Grade 9 for Asynchronous/Distance learninglevieagacer
 
Grade 7 - Lesson 1 - Microscope and Its Functions
Grade 7 - Lesson 1 - Microscope and Its FunctionsGrade 7 - Lesson 1 - Microscope and Its Functions
Grade 7 - Lesson 1 - Microscope and Its FunctionsOrtegaSyrineMay
 
Chemistry 5th semester paper 1st Notes.pdf
Chemistry 5th semester paper 1st Notes.pdfChemistry 5th semester paper 1st Notes.pdf
Chemistry 5th semester paper 1st Notes.pdfSumit Kumar yadav
 
Digital Dentistry.Digital Dentistryvv.pptx
Digital Dentistry.Digital Dentistryvv.pptxDigital Dentistry.Digital Dentistryvv.pptx
Digital Dentistry.Digital Dentistryvv.pptxMohamedFarag457087
 
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 bAsymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 bSérgio Sacani
 
Genetics and epigenetics of ADHD and comorbid conditions
Genetics and epigenetics of ADHD and comorbid conditionsGenetics and epigenetics of ADHD and comorbid conditions
Genetics and epigenetics of ADHD and comorbid conditionsbassianu17
 
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRL
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRLGwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRL
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRLkantirani197
 
CYTOGENETIC MAP................ ppt.pptx
CYTOGENETIC MAP................ ppt.pptxCYTOGENETIC MAP................ ppt.pptx
CYTOGENETIC MAP................ ppt.pptxSilpa
 

Recently uploaded (20)

Human genetics..........................pptx
Human genetics..........................pptxHuman genetics..........................pptx
Human genetics..........................pptx
 
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
Human & Veterinary Respiratory Physilogy_DR.E.Muralinath_Associate Professor....
 
Phenolics: types, biosynthesis and functions.
Phenolics: types, biosynthesis and functions.Phenolics: types, biosynthesis and functions.
Phenolics: types, biosynthesis and functions.
 
300003-World Science Day For Peace And Development.pptx
300003-World Science Day For Peace And Development.pptx300003-World Science Day For Peace And Development.pptx
300003-World Science Day For Peace And Development.pptx
 
Factory Acceptance Test( FAT).pptx .
Factory Acceptance Test( FAT).pptx       .Factory Acceptance Test( FAT).pptx       .
Factory Acceptance Test( FAT).pptx .
 
+971581248768>> SAFE AND ORIGINAL ABORTION PILLS FOR SALE IN DUBAI AND ABUDHA...
+971581248768>> SAFE AND ORIGINAL ABORTION PILLS FOR SALE IN DUBAI AND ABUDHA...+971581248768>> SAFE AND ORIGINAL ABORTION PILLS FOR SALE IN DUBAI AND ABUDHA...
+971581248768>> SAFE AND ORIGINAL ABORTION PILLS FOR SALE IN DUBAI AND ABUDHA...
 
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune Waterworlds
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune WaterworldsBiogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune Waterworlds
Biogenic Sulfur Gases as Biosignatures on Temperate Sub-Neptune Waterworlds
 
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry
GBSN - Biochemistry (Unit 2) Basic concept of organic chemistry
 
Clean In Place(CIP).pptx .
Clean In Place(CIP).pptx                 .Clean In Place(CIP).pptx                 .
Clean In Place(CIP).pptx .
 
biology HL practice questions IB BIOLOGY
biology HL practice questions IB BIOLOGYbiology HL practice questions IB BIOLOGY
biology HL practice questions IB BIOLOGY
 
Cyanide resistant respiration pathway.pptx
Cyanide resistant respiration pathway.pptxCyanide resistant respiration pathway.pptx
Cyanide resistant respiration pathway.pptx
 
LUNULARIA -features, morphology, anatomy ,reproduction etc.
LUNULARIA -features, morphology, anatomy ,reproduction etc.LUNULARIA -features, morphology, anatomy ,reproduction etc.
LUNULARIA -features, morphology, anatomy ,reproduction etc.
 
Module for Grade 9 for Asynchronous/Distance learning
Module for Grade 9 for Asynchronous/Distance learningModule for Grade 9 for Asynchronous/Distance learning
Module for Grade 9 for Asynchronous/Distance learning
 
Grade 7 - Lesson 1 - Microscope and Its Functions
Grade 7 - Lesson 1 - Microscope and Its FunctionsGrade 7 - Lesson 1 - Microscope and Its Functions
Grade 7 - Lesson 1 - Microscope and Its Functions
 
Chemistry 5th semester paper 1st Notes.pdf
Chemistry 5th semester paper 1st Notes.pdfChemistry 5th semester paper 1st Notes.pdf
Chemistry 5th semester paper 1st Notes.pdf
 
Digital Dentistry.Digital Dentistryvv.pptx
Digital Dentistry.Digital Dentistryvv.pptxDigital Dentistry.Digital Dentistryvv.pptx
Digital Dentistry.Digital Dentistryvv.pptx
 
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 bAsymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
 
Genetics and epigenetics of ADHD and comorbid conditions
Genetics and epigenetics of ADHD and comorbid conditionsGenetics and epigenetics of ADHD and comorbid conditions
Genetics and epigenetics of ADHD and comorbid conditions
 
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRL
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRLGwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRL
Gwalior ❤CALL GIRL 84099*07087 ❤CALL GIRLS IN Gwalior ESCORT SERVICE❤CALL GIRL
 
CYTOGENETIC MAP................ ppt.pptx
CYTOGENETIC MAP................ ppt.pptxCYTOGENETIC MAP................ ppt.pptx
CYTOGENETIC MAP................ ppt.pptx
 

DNA Sequencing- Sanger's Method

  • 1. DNA SEQUENCING SANGER’S METHOD HARSHA JOSEPH MSc LIFE SCIENCE
  • 2. What is sequencing? DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward.
  • 3. Sequencing an entire genome (all of an organism’s DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus." However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project.
  • 4. Sanger sequencing: The chain termination method INTRODUCTION • The DNA sequencing method developed by Fred Sanger forms the basis of automated "cycle" sequencing reactions today. • In the 1980s, two key developments allowed researchers to believe that sequencing the entire genome could be possible. • The first was a technique called polymerase chain reaction (PCR) that enabled many copies of DNA sequence to be quickly and accurately produced. • The second, an automated method of DNA sequencing, built upon the chemistry of PCR and the sequencing process developed by Frederick Sanger in 1977.
  • 5. • Sanger sequencing method of DNA sequencing was first commercialized by Applied Biosystems. • It was the most widely used sequencing method for approximately 40 years.
  • 6. • Regions of DNA up to about 900 base pairs in length are routinely sequenced using this method. • In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. • The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.
  • 7. Requirements for Sanger sequencing Fred Sanger’s method of DNA sequencing was based on Arthur Kornberg’s earlier work on DNA Replication. A new DNA strand is synthesized using an existing strand as a template.Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction. They include: • A DNA polymerase enzyme • A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase • The four DNA nucleotides (dATP, dTTP, dCTP, dGTP) • The template DNA to be sequenced However, a Sanger sequencing reaction also contains a unique ingredient: • Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye.
  • 8. Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a new nucleotide to be added to an existing chain. PRINCIPLE
  • 9. • The 5’ carbon of an “incoming” deoxynucleotide (dNTP) is joined to the 3’ carbon at the end of the chain. Hydroxyl groups in each position form ester linkages with a central phosphate. In this way, the nucleotide chain elongates. • The key to Sanger’s sequencing method is the peculiar chemistry of dideoxynucleotides (ddNTP). Like a deoxynucleotide, a ddNTP is incorporated into a chain by forming a phosphodiester linkage at its 5’ end. • However, the ddNTP lacks a 3’ hydroxyl group (OH) necessary to form the linkage with an incoming nucleotide. So the addition of a ddNTP halts elongation.
  • 10. Method • To sequence DNA, four separate reactions are necessary – one to provide sequence information about each of the nucleotides. Each reaction contains: template DNA, a short primer (about 20 nucleotides), DNA polymerase, and the four dNTPs (one radioactively labeled). • One type of ddNTP – A, T, C, or G – is added to each.
  • 11.
  • 12. The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the primer can bind to the single- stranded template.
  • 13. The DNA Polymerase makes no distinction between dNTPs or ddNTPs. Each time a ddNTP is incorporated, in this case ddATP, synthesis is “terminated” and a DNA strand of a discrete size is generated. In this sequencing example, the ddATP (purple) has terminated the reaction. The dATP happens to be the radioactive tracer, but this has no effect on elongation.
  • 14. Because billions of DNA molecules are present, the elongation reaction can be terminated at any adenine position. This results in collections of DNA strands of different lengths. The same is true for the other three terminator reactions.
  • 15. • Each reaction is then loaded into a separate lane of a polyacrylamide gel containing urea, which prevents the DNA strands from renaturing during electrophoesis. • Ionized phosphates give the DNA molecule a negative charge, so DNA migrate toward the positive pole of an electric field. The movement of DNA molecules through the polyacrylamide matrix is size dependent. • Over the course of electrophoresis, shorter DNA molecules will move further down the gel than larger ones. Millions of terminated molecules of the same size will migrate to the same place and “band” in the gel.
  • 16. After electrophoresis, the gel is sandwiched against X-ray film. The radioactive adenine in the synthesized DNA emit beta particles that expose the film, making a record of the positions of DNA bands in the gel. The sequencing gel is then read from bottom to top. The sequence of bands in the various terminator lanes gives the sequence of nucleotides in the template DNA.
  • 17. Another method for reading the sequence: • The process of replication is repeated in a number of cycles. By the time the cycling is complete, it’s virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA. The ends of the fragments will be labeled with dyes that indicate their final nucleotide.
  • 18. • After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis. • Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the “finish line” at the end of the tube, it’s illuminated by a laser, allowing the attached dye to be detected. • From the colors of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in fluorescence intensity. The DNA sequence is read from the peaks in the chromatogram.
  • 19.
  • 20. Uses and limitations • Sanger sequencing gives high-quality sequence for relatively long stretches of DNA (up to about 900 base pairs). It's typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR. • However, Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome or metagenome (the “collective genome” of a microbial community). For tasks such as these, new, large-scale sequencing techniques are faster and less expensive.