DNA sequencing Maxam and Gilbert, Sanger, Automated, Nextgen sequencing

1. DNA Sequencing
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. DNA Sequencing:
 DNA sequencing refers to methods for determining the order of the nucleotides bases adenine, guanine, cytosine and
thymine in a molecule of DNA.
 The first DNA sequence was obtained by academic researchers, using laboratories methods based on 2- dimensional
chromatography in the early 1970s.
 By the development of dye based sequencing method with automated analysis, DNA sequencing has become easier and
faster.

3. Methods of DNA sequencing:
Two main methods are widely known to be used to sequence
DNA:
1. The Chemical Method (also called the Maxam–Gilbert
method after its inventors). By using this method, they had
sequenced 24 nucleotides only. However, their method
was published after two years of Sanger’s method.
2. The Chain Termination Method (also known as the
Sanger dideoxy method after its inventor).
 Maxam–Gilbert technique depends on the relative chemical
liability of different nucleotide bonds, whereas the Sanger
method interrupts elongation of DNA sequences by
incorporating dideoxynucleotides into the sequences.
 The chain termination method is the method more usually
used because of its speed and simplicity.
Sequencing of an Oligonucleotide by Maxam-Gilbert method

4. 1. Chemical Cleavage Method (Maxam–Gilbert Method):
Maxam Gilbert sequencing is a method of DNA sequencing developed by
Allan Maxam and Walter Gilbert in 1976–1977. This method is based on
nucleobase-specific partial chemical modification of DNA and subsequent
cleavage at specific bases of the DNA backbone at sites adjacent to the
modified nucleotides.
 The method requires radioactive labelling at one end and purification of the
DNA fragment to be sequenced.
 Chemical treatment generates breaks at small proportions of one or two
of the four nucleotide based in each of four reactions (G, A+G, C, T +
C).
 Thus a series of labelled fragments is generated, from the radiolabelled end
to the first ‘cut’ site in each molecule.
 The fragments in the four reactions are arranged side by side in gel
electrophoresis for size separation.
 To visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each corresponding to a
radiolabelled DNA fragment, from which the sequence may be inferred.
Features:
 Base-specific cleavage of DNA by certain chemicals
 Four different chemicals, one for each base
 A set of DNA fragments of different sizes
 DNA fragments contain up to 500 nucleotides
 Hydrazine: T + C
 Hydrazine NaCl: C
 Dimethyl sulfate: A + G
 Piperidine: G
Sequencing of an Oligonucleotide by Maxam-Gilbert method

5. Procedure:
1. DNA extraction is the very first step. After that, the DNA is denatured using
the heat denaturation method and single-stranded DNA is generated.
2. The phosphate (5’P) end of the DNA is removed and labelled by the
radiolabeled P32. The enzyme named phosphatase removes the phosphate
from the DNA and simultaneously, the kinase adds the 32P to the 5’ end of it.
3. 4 different chemicals are used to cleave DNA at four different positions;
hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides
while dimethyl sulfate and piperidine attack purine nucleotides.
 Hydrazine: T + C
 Hydrazine NaCl: C
 Dimethyl sulfate: A + G
 Piperidine: G
4. An equal volume of 4 different ssDNA samples is taken into 4 different tubes
each containing 4 different chemicals. The samples are incubated for
sometimes and electrophoresed in polyacrylamide gel electrophoresis. The
results of the chemicals cleavage of four different tubes are shown in the
figure below.
5. Autoradiography is used to visualize the separation of DNA fragments. Due
to the radiolabelled 32P end of the DNA, the DNA bands visualized through
autoradiography.
Sequencing of an Oligonucleotide by Maxam-Gilbert method

6. Procedure:
1. DNA extraction is the very first step. After that, the DNA is denatured using
the heat denaturation method and single-stranded DNA is generated.
2. The phosphate (5’P) end of the DNA is removed and labelled by the
radiolabeled P32. The enzyme named phosphatase removes the phosphate
from the DNA and simultaneously, the kinase adds the 32P to the 5’ end of it.
3. 4 different chemicals are used to cleave DNA at four different positions;
hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides
while dimethyl sulfate and piperidine attack purine nucleotides.
 Hydrazine: C + T
 Hydrazine NaCl: C
 Dimethyl sulfate: A + G
 Piperidine: G
4. An equal volume of 4 different ssDNA samples is taken into 4 different tubes
each containing 4 different chemicals. The samples are incubated for
sometimes and electrophoresed in polyacrylamide gel electrophoresis. The
results of the chemicals cleavage of four different tubes are shown in the
figure below.
5. Autoradiography is used to visualize the separation of DNA fragments. Due
to the radiolabelled 32P end of the DNA, the DNA bands visualized through
autoradiography. Sequencing of an Oligonucleotide by Maxam-Gilbert method

7. Advantages:
 Purified DNA can be read directly
 Homopolymeric DNA runs are sequenced as efficiently as
heterogeneous DNA sequences
 Can be used to analyze DNA protein interactions (i.e.
footprinting)
 Can be used to analyze nucleic acid structure and epigenetic
modifications to DNA
Disadvantages:
 It requires extensive use of hazardous chemicals.
 It has a relatively complex set up / technical complexity.
 It is difficult to “scale up” and cannot be used to analyze
more than 500 base pairs.
 The read length decreases from incomplete cleavage
reactions.

8. 2. Sanger Sequencing- Dideoxy Chain terminator
method
Sanger sequencing, also known as the “chain
termination method”, is a method for determining the
nucleotide sequence of DNA.
The method was developed by two time Nobel
Laureate Frederick Sanger and his colleagues in
1977, hence the name the Sanger Sequence.
The method is also known as the first-generation
DNA sequencing method.
Sanger’s method of gene sequencing is also known as
dideoxy chain termination method.

9. Principle:
The key principle of the Sanger method was the use of
dideoxynucleotide triphosphates (ddNTPs) as DNA chain
terminators.
 A DNA primer is attached by hybridization to the template
strand and deoxynucleosides triphosphates (dNTPPs) are
sequentially added to the primer strand by DNA polymerase.
 The M13 primer is designed along with the known
sequences at 3’ end of the template strand.
 The reaction mixture also contains dideoxynucleoside
triphosphate (ddNTPs) along with usual dNTPs.
 If during replication, ddNTPs is incorporated instead of
usual dNTPs in the growing DNA strand then the
replication stops at that nucleotide.
 The ddNTPs are analogue of dNTPs
 ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot
make phosphodiester bond with nest nucleotide, thus terminates the
nucleotide chain
 Respective ddNTPs of dNTPs terminates chain at their respective site.
For example ddATP terminates at A site. Similarly ddCTP, ddGTP and
ddTTP terminates at C, G and T site respectively.

10. Sanger Sequencing Steps
There are three main steps to Sanger sequencing.
1. Template preparation: DNA Sequence for Chain
Termination PCR
2. Generation of nested set of labelled fragments
3. Size Separation by Gel Electrophoresis and gel reading
4. Gel Analysis & Determination of DNA Sequence

11. 1. Template preparation: DNA Sequence for Chain
Termination PCR
The DNA sequence of interest is used as a template for a special
type of PCR called chain-termination PCR.
Steps:
1. Copies of template strand to be sequenced must be
prepared with short known sequences at 3’ end of the
template strand.
2. A DNA primer (M13 SEQUENCING PRIMER) is essential to
initiate replication of template, so primer preparation of
known sequences at 3’end is always required.

12. 2. Generation of nested set of labelled fragments:
Steps:
 Copies of each template is divided into four batches and
each batch is used for different replication reaction.
 Copies of standard primer, normal dNTPs and DNA
polymerase I are used in all four batches.
 To synthesize fragments that terminates at A,
1. ddATP (can be radiolabelled) is added to the reaction
mixture to the batch I along with dATP, dTTP, dCTP and
dGTP,
2. standard primer and
3. DNA polymerase I.
 Similarly, to generate, all fragments that terminates at
C, G and T,
the respective ddNTPs i.e. ddCTP, ddGTP and ddTTP are added
respectively to different reaction mixture on different batch
along with usual dNTPs.

13. 3. Size Separation by Gel Electrophoresis and gel reading
Steps:
 The reaction mixture from four batches are loaded into
four different well on polyacrylamide gel and
electrophoresed.
 The autoradiogram of the gel is read to determine the
order of bases of complementary strand to that of
template strand.
 The band of shortest fragments is at the bottom of
autoradiogram so that the sequences of
complementary strand are read from bottom to top.
4. Gel Analysis & Determination of DNA Sequence
The last step simply involves reading the gel to determine the
sequence of the input DNA.
In manual Sanger sequencing, the user reads all four lanes of
the gel at once, moving bottom to top, using the lane to
determine the identity of the terminal ddNTP for each band. For
example, if the bottom band is found in the column corresponding
to ddGTP, then the smallest PCR fragment terminates with
ddGTP, and the first nucleotide from the 5’ end of the original
sequence has a guanine (G) base.

14. Significance of DNA Sequencing:
 Information obtained by DNA sequencing makes it possible to understand or
alter the function of genes.
 DNA sequence analysis demonstrates regulatory regions that control gene
expression and genetic “hot spots” particularly susceptible to mutation.
 Comparison of DNA sequences shows evolutionary relationships that provide
a framework for definite classification of microorganisms including viruses.
 Comparison of DNA sequences facilitates identification of conserved regions,
which are useful for development of specific hybridization probes to detect
microorganisms including viruses in clinical samples.
 DNA sequencing has become sufficiently fast and inexpensive to allow
laboratory determination of microbial sequences for identification of microbes.
Sequencing of the 16S ribosomal subunit can be used to identify specific
bacteria. Sequencing of viruses can be used to identify the virus and
distinguish different strains.
 DNA sequencing shows gene structure that helps research workers to find out
the structure of gene products.

15. Automated DNA sequencing:
 The manual Sanger method was tedious. However, recent
advancement into the sequencing makes it easy and rapid to use.
The semi-automated Sanger sequencing method is based on the
principle of Sanger’s method with some minor variations.
 Instead of the 4 different reactions, the automated DNA
sequencing carried out in the single tube and the DNA runs in a
single lane.
 Here, in the semi-automated DNA sequencing, the fluorescent-
labeled set of primers are used, instead of ddNTPs. Thus four
different primers give four different peaks.
 The PAGE method isn’t capable of separating all the fragments
in a single reaction. Therefore, alternatively, the capillary gel
electrophoresis method is practiced. This method separates each
and every single fragment precisely.
 The capillary electrophoresis used to separate DNA molecules
on the basis of the size, it is powerful enough to separate single
base pair fragment. The chromatogram generated through the
C.E sent the output as a fluorescent peak.
The advanced semi-automated Sanger sequencing
method is more accurate, reliable and faster than
the traditional method.

16. Three Basic Steps of Automated Sanger Sequencing
 The read capacity of the Sanger sequencing is higher as
compared with the chemical degradation method. It can
sequence 700 to 800bp sequence in a single run, therefore,
it is more suitable for sequencing bacterial or other
prokaryotic genomes.
 It is more advanced and automated. Even the error rate is
very low as compared with the conventional chain
termination method. Still, it is time-consuming and a high-
cost method.

17. Automated DNA sequencing

18. Next Generation Sequencing (NGS)
Important Next Generation Sequencing Techniques
The next-generation sequencing platform is different from the Sanger technique or chain
termination method of DNA sequencing. Broadly, it amplifies millions of copies of a
particular fragment in a massively parallel fashion and the “reads” are analyzed by the
computational program.
 Pyro sequencing
 Illumina (Solexa) sequencing
 Lynx therapeutics’ massively parallel signature sequencing (MPSS)
 Polony sequencing
 SOLiD sequencing
 DNA nanoball sequencing
 Helioscope single molecule sequencing
 Single molecule SMRT sequencing
 Single molecule real time (RNAP) sequencing
Next Generation Sequencing (NGS) is a powerful platform that has enabled the sequencing of
thousands to millions of DNA molecules simultaneously.

19. The generations of sequencing:
First Generation
 Maxam and Gilbert DNA sequencing and Sanger DNA Sequencing
Second Generation Sequencing
 Pyrosequencing
 Sequencing by Reversible Terminator Chemistry
 Sequencing by Ligation
Third Generation Sequencing
 Single Molecule Fluorescent Sequencing
 Single Molecule Real Time Sequencing
 Semiconductor Sequencing
 Nanopore Sequencing
Fourth Generation Sequencing
Aims conducting genomic analysis directly in the cell