There are 2 major methods for sequencing DNA

The enzymatic method developed by Sanger and the chemical method of Gilbert.

Both methods use a similar principle. They generate large numbers of oligonucleotides that begin at a fixed point and terminate at a specific type of base.

The sequencing reaction consists of 4 different reactions that are performed in parallel. There is one for each base (A, T, G, and C).

These reactions produce many fragments of different sizes and these are separated in a polyacrylamide gel that can resolve differences as small as 1 nucleotide.

After the 4 reactions are fractionated in the “sequencing gel” the order of nucleotides can be read directly from the gel.

The Sanger method uses controlled synthesis of DNA to generate a population of fragments that terminate at specific points.

Dideoxy nucleoside triphosphates (ddNTPs): nucleoside analogs that lack the OH group at positions 2 and 3 of the sugar. When incorporated into the growing DNA strand, the chain can grow no longer (there is no place for the next sugar to attach).

The sequencing reaction depends on chain termination

1.

Start with a primer that binds to one end of the DNA and is radioactively tagged. It allows you to visualize each band in a gel.

2.

Run 4 DNA synthesis reactions in separate tubes. Each reaction has a surplus of dATP, dTTP, dCTP, and dGTP.

3.

The first reaction also has a small amount of ddGTP, the second reaction has some ddATP, the third ddCTP, and the fourth ddTTP.

4.

In the first tube, the ddGTP stops one of the growing DNA molecules as soon as it is incorporated. Some DNAs grow a little longer before the DDGTP is incorporated, some are a little shorter.

The bottom line is that each tube contains a large population of DNAs of random and different sizes and all end in G.

5.

The same process occurs in the other 3 reactions with ddTTP, ddCTP, and ddATP.

6.

After each of the four reactions is complete, the contents are loaded into a well of a sequencing gel. This is a polyacrylamide gel that is capable of resolving 2 molecules that differ in length by only 1 base pair.

7.

The sequencing gel is dried and covered with an X-ray film to visualize radioactive bands.

8.

To obtain the DNA sequence, you start at the bottom of the gel with the smallest oligo. By counting each successively larger oligonucleotide you can see whether it ends in the G, C, T, or A reaction lanes.

Overview of Sanger sequencing method

A typical sequencing gel

This is an X-ray film that was exposed to P32 that was incorporated into the bands of a sequencing gel.

Pull out your magnifying glass and count each band starting at the bottom to determine the sequence It’s AAAAAACGGACCGGG etc.

What are sequencing reactions used for?

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To characterize newly cloned DNAs To confirm the identity of a mutation To check for gene polymorphisms To sequence genomes In the past, sequencing reactions were often performed in the lab on a small scale as needed.

Today, many large research labs have a core facility for sequencing. This is a lab that exists solely to sequence DNA for others for cash. It is much cheaper than trying to do the job yourself.

If your institution has no core facility for sequencing, the next best alternative is to send the DNA out to a biotechnology sequencing lab.

What about sequencing whole genomes?

Large scale sequencing is a different story. It is performed using labs with automated sequencers that work day and night These machines use the same basic reaction employing ddNTPs but Each reaction is tagged with colored fluorochrome rather than labeling the DNA with radioactive primers. As the newly synthesized DNA molecules are fractionated by size through the gel, they pass through a laser beam. The laser beam is counted by a photomultiplier tube which registers the order of yellow, red, green, or blue bands at the ends of each chain.

In the human genome sequencing project, large rooms filled with automatic sequencers work continually for months to years. In addition, the job of sequencing 3 billion base pairs is so large that it was broken up into a consortium. Many labs throughout the world are working to sequence different chromosomes.

The first draft of the human genome is finished and it contains about 30,000 genes.

Overview of automated sequencing

Maxam Gilbert sequencing: the chemical method

This method starts with a population of full length DNAs and chops them up at specific nucleotides. Think of it as a Sanger reaction going backwards.

1.

End label the DNA with P32 to visualize the fragments in a gel 2.

Set up 4 different reactions. Each reaction modifies one of the 4 bases on the DNA (A, T, C, and G) making it susceptible to cleavage. For example, dimethyl sulfate (DMS) methylates G and makes it a target for DNA strand cleavage. If you only use a little DMS, you can methylate and cleave strands randomly throughout the DNA molecule. As with the Sanger reaction, this produces a population of oligonucleotides that range in size and which all end in G.

3.

Perform 3 other chemical reactions in separate tubes to specifically modify and cleave DNA at each of the other 3 bases.

4.

Add each of the 4 reactions to a sequencing gel and fractionate by size. Read the sequence by looking at the X-ray film.

The Maxam Gilbert reaction is not often used for sequencing anymore because the enzymatic method of Sanger is easier and faster.