How does Sanger Sequencing Work? – Seq It Out #1

Let’s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. Yea, you guessed it! I am talking about Sanger Sequencing by capillary electrophoresis. Many might ask, “why is it called Sanger Sequencing?”

Sanger Sequencing is named after the inventor of this ground breaking technology, Dr. Frederick Sanger, who developed this method over 40 years ago in the mid-70s.

So, what are the basics of Sanger Sequencing?

It all starts by having a short primer binding next to the region of interest. In the presence of the 4 nucleotides, the polymerase will extend the primer by adding on the complementary nucleotide from the template DNA strand. To find the exact composition of the DNA sequence, we need to bring this reaction to a defined stop that allows us to identify the base of the very end of this particular DNA fragment. Sanger did this by removing an oxygen atom from the ribonucleotide. Such a nucleotide is called a dideoxynucleotide. This is analogous to throwing a wrench into a gear. The polymerase enzyme can no longer add normal nucleotides onto this DNA chain. The extension has stopped and we now need to identify what it is. We identify the chain terminating nucleotide by a specific fluorescent dye, 4 specific colors to be exact. Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end.

The extension products are then separated by Capillary Electrophoresis or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer. During CE, an electrical field is applied so that the negatively charged DNA fragments move toward the positive electrode. The speed at which a DNA fragment migrates through the medium is inversely proportional to its molecular weight. This process can separate the extension products by size at a resolution of one base. A laser excites the dye labeled DNA fragments as they pass through a tiny window at the end of the capillary. The excited dye emits a light at a characteristic wavelength that is detected by a light sensor. Software can then interpret the detected signal and translate it into a base call. When the sequencing reaction is performed in the presence of all four terminated nucleotides, you eventually get a pool of DNA fragments that are measured and separated base by base. What you will get in the end is a data file showing the sequence of the DNA in a colorful electropherogram and a text file which you can use to answer the questions you may be asking.

And that in a nutshell is Sanger Sequencing.

[ornellafera]

well-explained, short, comprising all steps = really nice video Thanks!!! :)

[zakriauthmani]

Thank you for this short and targeted approach to answer the the sanger for those who haven't been through it practically.

[retrostyledanish]

My goodness it really helped
Thankyou so much
I hope my exams would be better now

[chrisl.]

The clear writing and the awesome drawings made my day

[Cavers]

00:00 Brillinat opening!!! Oh hey I didn't see ya there!!!

[sciencetrainee]

Thank you so much for this video! I've been stuck on basics while reading articles. This time, one video and boom - the concept is clear! I feel ready-to-use Sanger Sequencing in my workplace :D

[crazyGBG]

I don't think I could have done my honours project without this channel.

[gronkmonster]

Thanks for the easy to understand explanation and graphics. Helped me understand a procedure that took me weirdly long to get the concept.

[laurelcook]

I understand the theory 100% and how it's supposed to work. However, I don't know how well this will work in real life since there's such a tiny size difference.

[leonemaledetto]

Oh this video Is JUST perfect, answered all my questions the day before the big test, Thanks Natalie!

[sarahziegel]

THIS VIDEO IS FIRE YAS YOY GET IT GURL THANK YOU SO MUCH BLESS UR HEART ANGEL

[faramund]

Love the effort but can you *show* us?
In other words, can this whole process be perceived under a microscope and then recorded for us to see?
Or can it really only be 'seen' by the laser?
If so can we at least see how the laser does this?
It would be really helpful in understanding what's going on.

[CanadianJenCJ]

Where can you buy a gold pipette? That is so cool.

[ProperJudgment]

this sequencing is insane if you think about it

[nobody]

Does this mean it can be used de novo ?

[bikashdas]

nice explained, i am great full to you . thank you

[md.shariqulreedoy]

I have a question. Here we have seen what are the dideoxynucleotides that are at the end of a DNA fragment? However, what about the identification of each nucleotides in a single DNA fragment?

[josveldscholte]

The DNA in the capillary should be drawn single stranded! Because only the new strands are labeled and the template strands might be huge or even not .bef homogeneously sized, doulble strand electrophoresis would give non-interpretable results!

[MasayoMusic]

How expensive is the equipment involved in Sanger Sequencing if I wanted to sequence my own DNA? Is it feasible for those that are not in the research field, but want to run their own experiments in a home-made lab? How about second-hand equipment? Thank you.

[TheJoshtheboss]

I still don't get how does the software know which ddNTP is the start of the sequence.
I get it will be one of the shortest fragments. But how do we know the shortest fragment is the 1st when it's random for all 4 ddNTPs