DNA SEQUENCING

TECHNIQUES
Presented By : Group 01
 CONTENTS

1. DNA Sequencing
2. Maxam & Gilbert Method
3. Sanger Method
4. Conclusion
5. References
 DNA SEQUENCING


DNA sequencing is the process of determining the precise order of nucleotides within a
DNA molecule. It includes any method or technology that is used to determine the order of
the four bases adenine, guanine, cytosine, and thymine in a strand of DNA.

DNA sequencing is used to identify mutations, diagnose diseases, and to study the
evolution of organisms. It is also used in criminal investigations to identify suspects, and in
gene therapy to diagnose genetic disorders.

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 MAXAM & GILBERT METHOD

INTRODUCTION: PRINCIPLES:

The Maxam-Gilbert sequencing method is a The Maxam-Gilbert sequencing method

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MAXAM & GILBERT

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STEPS:
Chemical Modification: The first step in the Separation: The cleaved fragments are then
Maxam-Gilbert sequencing method is the separated by gel electrophoresis. This
chemical modification of the DNA molecule. separates the fragments based on their size
This modification is achieved by treating the and charge.
DNA with various chemicals, such as nitrous Sequencing: The separated fragments are
acid or chemicals that contain sulfhydryl or then sequenced using radiolabeled
amine groups. nucleotides and autoradiography. The
Cleavage: The chemically modified DNA is sequence of the fragments can be determined
then subjected to specific conditions to by comparing the position of the radioactive
selectively cleave the phosphodiester bonds. bands on the autoradiogram with the known
This cleavage results in fragments of known size of the fragments.
length.

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ADVANTAGES: DISADVANTAGES:
High accuracy: The Maxam-Gilbert Complex and time-consuming: The
sequencing method provides high accuracy and Maxam-Gilbert sequencing method is a
is capable of detecting even small differences in complex and time-consuming process, and
the DNA sequence. requires specialized equipment and trained
personnel.
High throughput: This method is capable of
sequencing large amounts of DNA, making it Limited to a small number of samples: The
useful for large-scale sequencing projects. method is limited to a small number of
samples, as it is time-consuming and requires
Short sequences: The Maxam-Gilbert
a large amount of reagents.
sequencing method can be used to sequence
short fragments of DNA, making it useful for High cost: The cost of the reagents and
sequencing difficult targets such as repetitive equipment required for the Maxam-Gilbert
regions or genomic regions with high GC sequencing method can be high, making it
content. less accessible to some research groups.

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 SANGER METHOD

INTRODUCTION PRINCIPLE

The Sanger Method, also known as the dideoxy The Sanger Method is based on the
chain termination method, is a widely used termination of DNA synthesis by adding a
method for DNA sequencing. It was first small amount of chain-terminating
developed by Frederick Sanger in the 1970s and nucleotides (dideoxynucleotides) to the
was the first successful method for sequencing reaction mixture. These dideoxynucleotides
DNA. are analogues of regular nucleotides, but they
lack a 3'-hydroxyl group. This makes it
impossible for the DNA polymerase to add
another nucleotide to the growing strand,
resulting in the termination of DNA synthesis.

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FREDERICK SANGER

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PROCEDURE
Preparation of Template DNA: The first step Separation of DNA Fragments: The
in the Sanger Method is to prepare a single- reaction mixture is then subjected to gel
stranded template DNA of known length and electrophoresis, which separates the DNA
purity. fragments based on their size. The smaller
fragments will travel faster through the gel
Primer Annealing: A short complementary and will be separated from the longer
DNA primer is annealed to the single-stranded fragments.
template DNA.
Detection of DNA Fragments: The
Synthesis of DNA Fragments: DNA separated DNA fragments are then transferred
polymerase is added to the reaction mixture to a nitrocellulose or nylon membrane and
along with the four standard nucleotides (A, C,
hybridized with a labeled probe that is
G, T) and one of the four dideoxynucleotides
complementary to the primer. The resulting
(ddA, ddC, ddG, ddT). The reaction is
autoradiograph will show the positions of the
incubated, allowing the DNA polymerase to
terminations and will provide a visual
synthesize a new strand complementary to the
representation of the DNA sequence.
template. When a dideoxynucleotide is
incorporated into the growing strand, the chain 9
is terminated.
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ADVANTAGES LIMITATIONS
High Accuracy: The Sanger Method is a highly Low Throughput: The Sanger Method is a
accurate method for DNA sequencing, with relatively slow method for DNA sequencing,
error rates of less than 1%. with a limited throughput of around 1000
High Resolution: The Sanger Method provides base pairs per reaction.
high-resolution sequencing, allowing Cost: The Sanger Method is relatively
researchers to sequence individual bases of expensive compared to other DNA
DNA. sequencing methods.
Widely Used: The Sanger Method is widely Labor-Intensive: The Sanger Method is a
used in molecular biology research and has labor-intensive process, requiring a high level
been applied to a variety of applications, of technical skill and expertise to perform.
including the Human Genome Project.

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 CONCLUSION
In conclusion, DNA sequencing is a critical tool in modern biology that has revolutionized our
understanding of genetics, evolution, and disease. It has numerous applications, including the
diagnosis of genetic disorders, the study of evolution and population genetics, and the
development of personalized medicine. The rapid advancement in sequencing technology has
greatly reduced the cost and time required to sequence a genome, making it possible to sequence
large numbers of individuals at a relatively low cost. With its immense potential, DNA sequencing
is expected to continue playing a vital role in advancing our knowledge and understanding of the
living world.

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 REFERENCES

https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotec
hnology/a/dna-sequencing
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https://en.wikipedia.org/wiki/DNA_sequencing
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https://www.sciencedirect.com/topics/neuroscience/sanger-sequencing

https://www.cd-genomics.com/blog/sanger-sequencing-introduction-principle-and-protoco
l/

https://biotecharticles.com/Biotech-Research-Article/DNA-Sequencing-Maxam-Gilbert-Me
thod-285.html

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC392330/

https://microbenotes.com/dna-sequencing-maxam-gilbert-and-sanger-dideoxy-method/
THANK YOU