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Nucleotide Sequencing Compressed

DNA sequencing determines the order of nucleotide bases in DNA, with methods like Sanger sequencing and Maxam-Gilbert sequencing developed in the 1970s. Sanger sequencing involves amplifying DNA and using chain-terminating nucleotides to create fragments that are analyzed to determine the sequence, while Maxam-Gilbert sequencing relies on chemical reactions to cleave DNA at specific points. Both methods have their advantages and limitations, particularly in terms of efficiency and safety.

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16 views13 pages

Nucleotide Sequencing Compressed

DNA sequencing determines the order of nucleotide bases in DNA, with methods like Sanger sequencing and Maxam-Gilbert sequencing developed in the 1970s. Sanger sequencing involves amplifying DNA and using chain-terminating nucleotides to create fragments that are analyzed to determine the sequence, while Maxam-Gilbert sequencing relies on chemical reactions to cleave DNA at specific points. Both methods have their advantages and limitations, particularly in terms of efficiency and safety.

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anirudhballa1855
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DNA sequencing is the process of determining the sequence of nucleotide

bases (As, Ts, Cs, and Gs) in a piece of DNA.


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."
In the mid-1970s, two methods were developed to sequence DNA directly.
These were:
The Maxam–Gilbert sequencing method
The Sanger chain-termination method

In 1980, both Walter Gilbert and Frederick Sanger were awarded The Nobel
Prize in Chemistry for contributing to the determination of base sequences in
nucleic acids.
01
Regions of DNA up to about 900 base pairs in length are routinely sequenced using a method
called Sanger sequencing or the chain termination method.
Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977.
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 (PCR), which
copies DNA in vitro.

DNA polymerase enzyme


A primer
The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
The template DNA to be sequenced
Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP,
ddTTP, ddCTP, ddGTP), each labeled with a different color of dye
The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA
nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well
The mixture is first heated to denature the template DNA then cooled so that the primer can bind to
the single-stranded template.
Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize
new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain
until it happens to add a dideoxy nucleotide instead of a normal one.
At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.
This process 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.
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.
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.
The smallest fragment (ending just one nucleotide
after the primer) crosses the finish line first, followed
by the next-smallest fragment (ending two
nucleotides after the primer), and so forth
Thus, 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, as shown in the
chromatogram above. The DNA sequence is read from
the peaks in the chromatogram.
High-quality sequence for Expensive
relatively long stretches of Inefficient for larger-scale
DNA projects, such as the sequencing
Typically used to sequence of an entire genome or
individual pieces of DNA, metagenome
such as bacterial plasmids or
DNA copied in PCR.
02
Maxam–Gilbert sequencing is also known as chemical sequencing because chemical
reactions, rather than DNA and RNA amplification, are the basis of the method.

It’s quite easy to understand, though, and proceeds in just 4 steps:


Preparation of Your Sample (single-stranded chains and radiolabeled on the 5′ end,
usually with 32P.)
Cleavage
Electrophoresis and Autoradiography
Reading the Sequence
The DNA used in Maxam-Gilbert sequencing is first denatured into single-stranded chains and radiolabeled
on the 5′ end, usually with 32P.
The next step cleaves the DNA.. By taking advantage of piperidine and two chemicals that selectively attack
purines and pyrimidines (dimethyl sulfate and hydrazine, respectively), the DNA is cleaved at specific
points.
using different combinations of these chemicals, you can cleave a DNA sequence wherever there is a C,
wherever there is a C or a T; wherever there is a G, or wherever there is a G or an A.
These reactions are then loaded onto a high-percentage agarose gel to differentiate fragment sizes. The
fragments are visualized via the radioactive tag.
To read the sequence, you begin with the smaller fragments at the bottom of the gel. “Calling” each base
involves interpreting the band pattern relative to the four chemical reactions.
For example, if a band in the DNA sequence appears in both the G-reaction and the G+A-reaction lanes,
then that nucleotide is a G.
If a band in the DNA sequence appears only in the G+A-reaction lane, then it is an A.
Sequences are confirmed by running replicate reactions on the same gel and comparing the
autoradiographic patterns between replicates.
DNA Footprinting Its slow
Identifying DNA Uses Dangerous Chemicals
Modifications Comparatively Information-
Structural DNA Analysis Poor

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