3. INTRODUCTION:
What is DNA sequencing?
DNA sequencing determines the order of the four chemical building blocks - called "bases" - that make up the
DNA molecule.
Why is DNA sequencing Important?
All the Information required for the growth and development of an organism is encoded in DNA of its
Genome. So, DNA sequencing is fundamental to genome analysis and understanding the biological process
in general
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4. Methods:
To determine the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of
DNA two methods were used
1. Sanger; Chain termination sequencing method
2. Maxam and Gilbert; Chemical Sequencing are based on these methods
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5. Sanger sequencing 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 Frederick Sanger and his colleagues in
1977, hence the name the Sanger Sequencing.
• It is PCR based method.
• A modified DNA replication reaction.
• Growing chain are terminated by di-deoxynucleotidetriphosphates (ddNTP)
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6. Sanger sequencing Method:
Requirement for Sanger sequencing
• A DNA polymerase enzyme
• A primer acts as a "starter" for the DNA polymerase •
The four DNA nucleotides (dATP, dTTP, dCTP, dGTP) •
The template DNA to be sequenced .
• Dideoxy nucleotide (dd), or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each
labeled with a different color of dye
Dideoxy nucleotides 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. Once a dideoxynucleotide has
been added to the chain, there is no hydroxyl available and no further nucleotides can be added. The chain ends with
the dideoxynucleotide, which is marked with a particular color of dye depending on the base (A, T, C, or G) that it
carries. normal deoxynucleotide triphosphates (dNTPs) modified di-deoxynucleotide triphosphates (ddNTPs),
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8. Sanger sequencing Method:
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• Four separate reactions are needed in this process to test all four ddNTPs.
• The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly
read off the X-ray film or gel image.
9. Sanger sequencing Method:
Application;
Sanger sequencing was used in the HumanGenome Project to determine the sequences of
relatively small fragments of human DNA.
Sanger DNA sequencing is widely used for research purposes like
(1) targeting smaller genomic regions in a larger number of samples,
(2) sequencing of variable regions,
(3) verifying plasmid sequences, inserts, mutations,,
(4) identifying single disease-causing genetic variants.
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10. Sanger sequencing Method:
Advantages:
Sanger sequencing gives high-quality sequences 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.
Allows scientists to determine genome sequence
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11. Sanger sequencing Method:
Disadvantages:
.
Sanger sequencing is expensive and inefficient for larger-scale projects, such as the
sequencing of an entire genome. For tasks such as these, new, large-scale sequencing
techniques are faster and less expensive.
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