in this video we're going to look at how to sequence DNA. how to sequence DNA?
why would i want to do that? what did DNA ever do for me? what did DNA ever do for you? are you
kidding?! DNA is the molecule that carries your genetic instructions for development
functioning growth and reproduction. And it's not just you, it's for all living organisms.
What does it look like? well thanks to a technique of electron microscopy and x-ray crystallography
we were able to visualize it in 1950s - the structure of DNA that is - in 1950s the structure
and what its function is was uncovered by James Watson Francis Crick and Rosalind Franklin.
DNA is composed of two chains that are coiled around each other to form what is called a double
helix. the chains are made from individual units called nucleotides - kind of like how amino acids
make up proteins. for DNA the chain the individual units are nucleotides the nucleotides come in four
flavors you have a sugar called deoxyribose; this is where the name deoxyribonucleic acid
the longer form of DNA actually comes from is this sugar that's a component of the structure
so the deoxyribose are nitrogenous base along with a phosphate group make up a nucleotide
making new DNA involves an enzyme called DNA polymerase coming along and copying the existing
dna it makes new dna by joining repeating units of one of four bases adenine cytosine guanine and
thymine so how does it join them together? this is crucial this part is really important because
this is how you are able to synthesise DNA or sequence DNA in the lab the critical parts of the
structure of DNA that make this possible are the three phosphates that are on the what's referred
to as the 5-prime end or the 5-prime portion of the structure and then the hydroxyl groups at the
3-prime end of the DNA of any nucleotide the triphosphates provide energy and they get joined
to the existing nucleotide of an existing DNA template at the 3-prime hydroxyl side so at the
hydroxyl side the phosphate gets joined and one phosphate of the triphosphate gets joined and two
are released and in this way you you grow the DNA template or sequence the DNA template by
just joining each new one to the existing one at the hydroxyl end the 3-prime hydroxyl end and then
you use the phosphate of the new nucleotide to do this that's what allows you to link it you know
it's like this linking process of the phosphates from the new nucleotides, to the hydroxyl of the
existing; the phosphate of the new nucleotide to the hydroxyl of the existing; the phosphate of the
new nucleotide to the hydroxyl of the existing one, and in this way you structure DNA or you
sequence new DNA. so one technique for doing this that existed the very first technique for
doing this is the Sanger termination method. Sanger - Fred Sanger - he had already been
involved in synthesising proteins so he had that experience and he came up with this method where,
knowing that you need the phosphate groups and you need a hydroxyl point in order to extend the DNA,
he came up with a clever idea of; what if we throw into the reaction mixture some nucleotides
(deoxyribonucleotides) that have the hydroxyl side of the structure altered so that it cannot
be extended. so whenever you incorporate one of those nucleotides where the hydroxyl site has been
messed with - so that it doesn't allow the next phosphate of the new one to be adjoined - then it
stops the reaction and if you stop the reaction a different part and then separate the DNA,
you're going to get different sequence.. different lengths all along and what's critical here too is
your primer; so you would have had a primer DNA doesn't get synthesised without its short little
piece that the polymerase needs to extend, so if you know the primer sequence then you
will be able to track the orientation of the new DNA that's being synthesised in the sense that,
i know my primer sequence and then i know after the primer sequence there's an Adenine
for example and then there was another Adenine then there was a cytosine for example then there
was a thymine then there was a guanine... see so in this way you can track how it's being added
and it reports to you what sequences are in the the unknown DNA that you want to sequence.
in addition to this altered hydroxyl part of any nucleotide the few nucleotides that
you use to sequence, he also has the idea of making it fluorescent. So then you can..
it will be a reporter to you when this incorporation happens. You will see that is
fluorescent. The Sanger method was actually what was used to sequence
the human genome as part of the human genome project back in the 1990s the human genome
project was started in the late 80s to put together a map of a human being. Any guesses
as to how long it took to put together a map of a human being? Let me know in the comments.
However with time there has been new methods for sequencing DNA
that are much faster. This is because they do lots of reactions at any one time. we
will get into that in part two of the video so stay tuned. thank you for watching go ahead
and hit the subscribe button to join the study group as we cover more of how DNA
sequencing is performed in the laboratory. i'll catch you in the next video, God bless.