{\rtf1\ansi\ansicpg1252\deff0\deflang1033{\fonttbl}
\viewkind4\uc1\pard\fs20 So okay guys in this video we will be talking
about next-generation sequencing.
And one of the tide of next-generation sequencing is ion torrent genome sequencing machine and
a 454 sequencing so sequencing method.
So be talking about the shotgun genome sequencing.
As we have a large DNA
we need to break it down into smaller fragments so we just use physical sharing method to
break those gene down and then sequence those genes.
Once we have the small fragments in hand the second task is to seek resource and analyse
the sequence results.
We analyse the sequence for the overlapping regions and then we get the sequence of the
long DNA or genome.
So let's begin with it.
So once we have the small fragment of DNA we added adductor molecules to both the terminal
of that DNA.
Those that adaptors are made by us so we know that adaptor sequence.
Not only adapter but he also added a barcode to warn of
the terminal of the gene.
It's very important to mark the genome with fluorescent dye so that we can track the position
of the DNA.
Once we have that they add that DNA to be actors to be the be consisting some adapter
complement sequences.
These are also produced synthetically in laboratory so the only those that are present in beads
are also of all known sequence.
The sequence in those adapter is complimented the to adaptors present in the DNA of or interest.
This whole system is automated and handled by robots.
The next stage is to amplify our gene of interest and usually we achieve their task in molecular
biology using PCR.
In this case of next-generation sequencing we will be using a bridge amplification method
for amplifying our gene of interest.
As the target DNA is very low you cannot get the target DNA
from the organism so we need to use some of the amplification method.
The mode of amplification is slightly different hear.
So once the DNA of interest with the help of adapter at such a sweet the bead with oligo
and the use the oligo as the primer and the gene of interest
as a template to produce more and more copy of that same gene.
And those genes which are not bound are washed away.
After that we take the beads along with the virgin of interest and put it into the micro
wells in tiny slides to carry out the sequencing reaction.
Once we put those nucleotides sequence is into the slide, that surely DNA sequencing
process can begin.
So we have a gene to be sequenced and a primer attached with it.
Then we provide only polymerase one nucleotide at a time into the wells.
We added the primer during the hybridisation process.
So the three-time hydroxyl group is free for the polymerisation.
And then we start adding nucleotides or one at a time
and those nucleotides are fluorescently labeled.
So every time or nucleotide is added in response to
the DNA sequence of
interest to fluorescence will be released and that fluoresce can be detected
with the detector attached with a computer.
That fluorescence will give us a peak and the height of the peak is proportional to
the number of
nucleotides
added with the gene of interest during sequencing.
\par \f1\par
}