DNA Sequencing
Principles of DNA Sequencing
DNA sequencing refers to the general laboratory technique for determining the exact
sequence of nucleotides, or bases, in a DNA molecule.
The sequence of the bases (often referred to by the first letters of their chemical names:
A, T, C, and G) encodes the biological information that cells use to develop and operate.
Establishing the sequence of DNA is key to understanding the function of genes and other
parts of the genome.
24 years after the discovery of the structure of DNA, two separate methods for
sequencing DNA were developed: the chain termination method and the chemical
degradation method. Both methods were equally popular to begin with, but, for many
reasons, the chain termination method is the method more commonly used today. This
method is based on the principle that single-stranded DNA molecules that differ in length
by just a single nucleotide can be separated from one another using polyacrylamide gel
electrophoresis.
There are several different methods available for DNA sequencing, each with its own
characteristics, and the development of additional methods represents an active area of
genomics research.
Sanger Sequencing
Sanger sequencing, also known as the “chain termination method,” was developed by the
English biochemist Frederick Sanger and his colleagues in 1977.
This method is designed for determining the sequence of nucleotide bases in a piece of
DNA (commonly less than 1,000 bp in length).
Sanger sequencing with 99.99% base accuracy is considered the “gold standard” for
validating DNA sequences, including those already sequenced through next-generation
sequencing (NGS).
Sanger sequencing was used in the Human Genome Project to determine the sequences of
relatively small fragments of human DNA (900 bp or less).
These fragments were used to assemble larger DNA fragments and, eventually, entire
chromosomes.
Principle of Sanger Sequencing
1. In Sanger sequencing, a DNA primer complementary to the template DNA (the DNA to
be sequenced) is used to be a starting point for DNA synthesis.
2. The polymerase extends the primer by adding the complementary dNTP to the template
DNA strand in the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C,
and T).
3. To determine which nucleotide is incorporated into the chain of nucleotides, four
dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) labeled
with a distinct fluorescent dye are used to terminate the synthesis reaction.
, 4. Compared to dNTPs, ddNTPs has an oxygen atom removed from the ribonucleotide,
hence cannot form a link with the next nucleotide.
5. Following synthesis, the reaction products are loaded into four lanes of a single gel and
subjected to gel electrophoresis.
6. The sequence of the DNA is determined according to the sizes of the separated
fragments.
Principles of DNA Sequencing
DNA sequencing refers to the general laboratory technique for determining the exact
sequence of nucleotides, or bases, in a DNA molecule.
The sequence of the bases (often referred to by the first letters of their chemical names:
A, T, C, and G) encodes the biological information that cells use to develop and operate.
Establishing the sequence of DNA is key to understanding the function of genes and other
parts of the genome.
24 years after the discovery of the structure of DNA, two separate methods for
sequencing DNA were developed: the chain termination method and the chemical
degradation method. Both methods were equally popular to begin with, but, for many
reasons, the chain termination method is the method more commonly used today. This
method is based on the principle that single-stranded DNA molecules that differ in length
by just a single nucleotide can be separated from one another using polyacrylamide gel
electrophoresis.
There are several different methods available for DNA sequencing, each with its own
characteristics, and the development of additional methods represents an active area of
genomics research.
Sanger Sequencing
Sanger sequencing, also known as the “chain termination method,” was developed by the
English biochemist Frederick Sanger and his colleagues in 1977.
This method is designed for determining the sequence of nucleotide bases in a piece of
DNA (commonly less than 1,000 bp in length).
Sanger sequencing with 99.99% base accuracy is considered the “gold standard” for
validating DNA sequences, including those already sequenced through next-generation
sequencing (NGS).
Sanger sequencing was used in the Human Genome Project to determine the sequences of
relatively small fragments of human DNA (900 bp or less).
These fragments were used to assemble larger DNA fragments and, eventually, entire
chromosomes.
Principle of Sanger Sequencing
1. In Sanger sequencing, a DNA primer complementary to the template DNA (the DNA to
be sequenced) is used to be a starting point for DNA synthesis.
2. The polymerase extends the primer by adding the complementary dNTP to the template
DNA strand in the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C,
and T).
3. To determine which nucleotide is incorporated into the chain of nucleotides, four
dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) labeled
with a distinct fluorescent dye are used to terminate the synthesis reaction.
, 4. Compared to dNTPs, ddNTPs has an oxygen atom removed from the ribonucleotide,
hence cannot form a link with the next nucleotide.
5. Following synthesis, the reaction products are loaded into four lanes of a single gel and
subjected to gel electrophoresis.
6. The sequence of the DNA is determined according to the sizes of the separated
fragments.
