Msc BT 202
Dr. Prerona B
Sanger sequencing
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.
https://www.youtube.com/watch?v=KTstRrDTmWI
https://www.youtube.com/watch?v=FvHRio1yyhQ
Next generation sequencing
Next-generation sequencing is the term applied to a variety of methods
that enable thousands or millions of DNA fragments to be sequenced in
parallel in a single experiment. The preparation and use of this
sequencing library is the distinctive feature that distinguishes these
methods from chain-termination sequencing, which is able to
sequence only individual DNA fragments, each fragment obtained by a
different PCR or from a different clone.
The common feature of the various next-generation sequencing methods is the
prior preparation of a library of DNA fragments that have been immobilized on
a solid support in such a way that multiple sequencing reactions can be carried
out side by side in a massively parallel array format (Figure 4.7). The fragments
are usually 100–500 bp in length, the precise sizes depending on the lengths of
the individual sequences that can be obtained by the next-generation method
that is being employed.
Most popular way of breaking genomic DNA down into fragments of these
sizes is by sonication, a technique that uses high-frequency sound waves to
make random cuts in DNA molecules.
Two different immobilization methods are commonly used in next-generation
sequencing. In the first method, the solid support is a glass slide that has been
coated with many copies of a short oligonucleotide. Adaptors, short pieces of
double-stranded DNA whose sequences match that of the oligonucleotide, are
ligated to the ends of the DNA fragments, which are then denatured.
The resulting single-stranded molecules attach to the glass slide by base
pairing between their adaptor sequences and the immobilized
oligonucleotides
In the second immobilization method, the solid support is provided by small
metallic beads that are coated with the protein streptavidin. Once again, the
DNA fragments are ligated to adaptors, in this case adaptors that carry a biotin
label attached to their 5ʹ-ends. Biotin is a small organic molecule that binds
strongly to streptavidin, so the fragments become attached to the metallic
beads by biotin– streptavidin linkages.
The ratio of DNA fragments to beads is set so that, on average, just one
fragment becomes attached to each bead. The beads are then shaken in an oil–
water mixture in order to generate an emulsion, with the conditions set so that
there will be just one bead in each aqueous droplet within the emulsion. Each
aqueous droplet is then transferred into a different well in a multiple array on a
plastic strip
The final step in preparation of the library is amplification of the immobilized
DNA fragments by PCR, to produce a sufficient number of identical copies to be
sequenced.
https://www.youtube.com/watch?v=jFCD8Q6qSTM
https://www.youtube.com/watch?v=-kTcFZxP6kM
Pyrosequencing
In this method, the reaction mixture
contains only deoxynucleotides, and
template copying proceeds without
artificial termination. Strand
synthesis is followed by detecting
flashes of chemiluminescence
generated by the enzyme sulfurylase
from the molecule of pyrophosphate
that is released each time DNA
polymerase adds a deoxynucleotide
to the 3ʹ-end of the growing strand.
Illumina sequencing
Currently the most popular method is based on reversible
terminator sequencing which, like the chain-termination
method, makes use of modified nucleotides that block
strand synthesis when incorporated at the end of a
polynucleotide that is being synthesized by a DNA
polymerase. The difference is that the termination step is
reversible, because the chemical group attached to the 3ʹ-
carbon of the modified nucleotide can be removed once
the identity of this nucleotide has been confirmed.
This removable blocking group is a fluorescent label, a
different one for each of the four nucleotides. There are no
normal deoxynucleotides present in the reaction mixture,
so each step in strand synthesis is accompanied by a pause,
during which an optical device detects the fluorescent
label, thereby identifying the terminal nucleotide
sequencing by oligonucleotide ligation and
detection (SOLiD)
The sequence is deduced not by
polymerase-directed synthesis of a new
DNA strand but by hybridization of a series
of oligonucleotides whose sequences are
complementary to that of the template.
Each of these oligonucleotides is labeled
with a fluorescent marker, but only four
different markers are used in total. This
means that the markers divide the 1024
oligonucleotides into four families of 256
sequences.
Ion semiconductor sequencing
The ion torrent method uses a similar approach to
pyrosequencing, with a repetitive series of
nucleotides flowed over an immobilized fragment
library.
However, with this method, the detection system is
directed at the hydrogen ions that, along with
pyrophosphate, are released every time a
nucleotide is incorporated into the growing strand.
The reaction is carried out with DNA fragments
immobilized on acrylamide beads, each bead in a
well lined with an ion-sensitive field effect
transistor (ISFET).
The ISFET generates an electronic pulse each time
it detects hydrogen ions, these pulses being related
to the flow of nucleotides over the well in order to
deduce the sequence of the immobilized
fragments. Read lengths of up to 400 bp are
possible
