DNA Sequencing; The convergence of four technologies

I. Organic Chemistry.

Everytime a new biological molecule is found, organic chemists begin making every possible modification. Many of these have been used as drugs to fight AIDS (e.g. AZT or ddI). One such modification of nucleotides were dideoxy nucleotides (ddNTPs).

II. Cloning.

III. In vitro DNA Synthesis.

Given a DNA template, an appropriate primer, DNA Polymerase I and the four dNTPs, it is possible to create a new strand
(5'----->3') of complementary DNA in vitro.

IV. Electrophoretic Fractionation.

Fragments of DNA can be fractionated in an electric field by the technique of electrophoresis. Because the charge density of the fragments are the same, they will fractionate based on the friction provided by the polysaccharide matrix in the agarose gel. Smaller fragments will migrate with more mobility than larger fragments.

To sequence DNA that has been cloned, just do as follows:

Cloned Insert

3'---------------------AGCTCTTGAAGTC-5'

5'--------------------3'Primer

1. Set up 4 tubes.

• To all tubes add:
  • cloned insert
  • primer
  • DNA Polymerase I
  • 4 dNTPs

2. To each of the four tubes add one ddNTP. Because they lack a 3'-OH, they terminate the polymerization process. View the animation demonstrating chain termination at this site [local].

3. Incubate and allow polymerase to function.

4. Load onto electrophoresis gel--one tube per lane--and fractionate by size.

5. Read the DNA sequence from the electrophoresis gel. The lane indicated by the letter (see figure) will contain fragments that were terminated with the appropriate ddNTP, thus identifying that nucleotide in the sequence.

See if you can draw the expected pattern of DNA fragments on the hypothetical gel below.

This method, using the dideoxynucleotides, was developed by F. Sanger who also developed the technology for sequencing proteins. There is a second technology that relies on differential reactivity of the four DNA bases that you may also encounter. This method is called the Maxam-Gilbert Method or "chemical sequencing". Much of this technology is now automated. Also, rather than running separate reactions with different individual ddNTPs, the technology employs ddNTPs with different fluorophores. These can be run in a single lane of an electrophoresis gel and scanned by a laser at the bottom of the gel. Such technology is shown in this animation.