Sanger Sequencing

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From the course by McMaster University

DNA Decoded

79 ratings

McMaster University

DNA Decoded

79 ratings

Are you a living creature? Then, congratulations! You’ve got DNA. But how much do you really know about the microscopic molecules that make you unique? Why is DNA called the “blueprint of life”? What is a “DNA fingerprint”? How do scientists clone DNA? What can DNA teach you about your family history? Are Genetically Modified Organisms (GMOs) safe? Is it possible to revive dinosaurs by cloning their DNA? DNA Decoded answers these questions and more. If you’re curious about DNA, join Felicia Vulcu and Caitlin Mullarkey, two biochemists from McMaster University, as they explore the structure of DNA, how scientists cracked the genetic code, and what our DNA can tell us about ourselves. Along the way, you’ll learn about the practical techniques that scientists use to analyze our genetic risks, to manipulate DNA, and to develop new treatments for a range of different diseases. Then, step into our virtual lab to perform your own forensic DNA analysis of samples from a crime scene and solve a murder.

From the lesson

DNA and Me

What does your DNA say about you? You may have heard that you share 99% of your genetic material with everyone else on the planet. That's true, but this week we're going to take a look at the less than 1% that makes you unique. We'll kick off by discussing the Human Genome Project, the massive scientific collaboration that mapped out the sequence of all our genetic material. The technologies developed while mapping out the human genome ushered in a new age in DNA research. Now, genome-wide association studies can analyze massive amounts of data, searching for genetic variations that are associated with particular diseases. Pharmacogenomics may help determine which drugs are likely be most effective for you. Genetic genealogy tests (such as Ancestry DNA, 23 and Me, and Family Tree DNA) will allow you to trace random mutations in your DNA that can provide clues to your ethnic heritage. As an added bonus, we'll discuss whether it would be possible to revive extinct species by studying ancient DNA.

DNA: Our Genetic Blueprint4:27

Sanger Sequencing3:20

The Human Genome Project5:08

Mammoths, Mummies, and Microbes: Ancient DNA10:52

Me and My Family Tree8:16

DNA and My Health5:20

That's a wrap!5:06

Meet the Instructors

Caitlin Mullarkey
Assistant Professor
Health Sciences
Felicia Vulcu
Assistant Professor
Biochemistry and Biomedical Sciences

[Music]

[Caitlin:] You might be asking, "How do scientists read the sequence of base pairs in DNA?"

[Felicia:] Good question!

[Caitlin:] We use a process called Sanger sequencing,

in honour of the scientist who pioneered the technique, Frederick Sanger.

Sanger was an amazing scientist who received two Nobel prizes.

His first Nobel, in 1958, was for his work on understanding the structure of proteins.

But more to the point here,

in 1980, he shared a Nobel prize with

two other researchers for figuring out a way to sequence DNA.

The process is both brilliant and simple,

as elegant solutions in science often are.

You may remember that in Week 2,

Felicia cooked up some PCR samples in her thermocycler

so we could identify the DNA of Dr Earl N. Myer's killer.

Well, DNA sequencing is a very similar process.

First, we use PCR to make many, many copies

of our DNA sample with all of the same base pairs of sequence.

[Felicia:] The heat and unzips the strands of DNA.

The primer marks the spot where we'll start to copy the DNA strand.

Taq DNA polymerase starts at the location of the DNA primer and

begins adding bases one at a time, according to Watson-Crick rules:

A pairs with T, C pairs with G.

Eventually, the DNA polymerase will add a base that puts the brakes

on the duplication process: a ddNTP.

[Caitlin:] ddNTPs come in four different flavours: A, T, C, and G.

Just like the regular bases.

Here's the genius part:

The ddNTPs have been specially treated,

so they light up in different colours.

The colours let you know where the end of the strand is.

Even better, the colour can tell you which base is last on this fragment of DNA.

You put many strands of DNA in the test tube.

So, there are many reactions going on in there.

Different strands will stop duplicating at different places.

So, the newly-replicated DNA will have many different lengths.

If you could take a quick look into the tube,

you might see something like this...

Now, we just need to sort these DNA fragments,

based on their size, to see each base in sequence.

[Felicia:] We know how to do this already.

We can use gel electrophoresis -- with a minor twist.

Instead of a giant slab of gel,

we have a very thin capillary tube with gel.

As the sample runs through this thin tube,

the molecular sieve sorts the DNA fragments out by their length.

The colours of each ddNTP associated with a different termination length of DNA

is detected using the colour properties of each ddNTP.

[Caitlin:] The computer output is in the form of these colourful peaks called a chromatogram.

Each peak corresponds to a different colour assigned to each of the four ddNTPs.

Also, each peak is detected at a specific length of the DNA.

So, all you have to do now is read the peaks from

left to right in order to figure out the sequence of DNA.

In this case, we have AGTCAGTC.

[Felicia:] So, next time you see one of these images on TV,

you'll know exactly what it means.

That Sanger guy and his pals were brilliant!

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