Locating Genes With Recombinant DNA Techniques

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This image illustrates the Sanger Technique of Radioactive Sequencing. Image by Abizar

The Sanger Technique (or ‘Chain Termination’ Method)

The Sanger technique relies on lining up increasingly longer DNA segments that commence at the same point and end with a known nitrogenous base. Scientists can consequently determine the positions of this particular base along the sequence by counting how many base lengths it is from the beginning of the strand.

The Sanger technique combines a small amount of ddNTP (di-deoxy) nucleotides with DNA polymerase, a single strand of the unknown DNA and normal nucleotides containing adenine, thymine, cytosine and guanine. DNA polymerase, an enzyme that promotes DNA chain elongation, proceeds to add nucleotides to the template strands, but the chain terminates whenever a ddNTP is randomly added.

This random chain termination arises because ddNTPs are lacking in a terminal hydroxyl group, which results in the inhibition of chain extension. Researchers run the DNA segments from all four reaction mixtures in separate lanes through an electrophoresis gel and deposit them on the gel in order of increasing chain length. They then determine the base sequence of the original DNA strand by reading off the marked probes from the shortest to the longest segment.

Recent Automated Gene Sequencing Methods

The Human Genome Project used the Sanger technique, coupled with the Celera ‘shotgun’ technique of shattering longer lengths of DNA into fragments for sequencing. In the last stage of the project, a faster version of this process emerged in which researchers placed all four ddNTPs in the same reaction mixture, and analyzed the resulting DNA sequences using a laser beam that transferred its data to a chromatogram.

This method could in fact analyse 24 samples at a time, significantly reducing the time and cost of DNA sequencing. Science has since developed further techniques that do not rely on a gel matrix for sequencing; these include scanning microscopes, mass spectrometry and flow-cytometry.

Pyrosequencing: New DNA Sequencing Process

Moreover, a new sequencing process known as Pyrosequencing may achieve a ten-fold reduction in cost and a 20-fold reduction in time compared to current Sanger techniques. As in the Sanger process, this method uses DNA polymerase to add complementary nucleotides to a single strand of the unknown DNA. The nucleotides, however, are not chain-terminating.

Researchers add each of the four different nucleotides in turn to the single-stranded DNA. When the correct nucleotide bonds to its complementary base on the template strand, it emits a photon of light, which the process records on a pyrogram.

New Developments in Genome Mapping

A pyrogram measure light emitted during pyrosequencing. Image by Zaimon

Although pyrosequencing still has some glitches, it promises to radically reduce sequencing time and cost in the future, to the point where individuals may soon be able to pay for their own genome mapping.

Other advances in gene sequencing, including new hybridization techniques, nanopore sequencing and ion detection systems, are now either under patent or in various stages of development.

Resources

DNA From the Beginnning. Alfred Henry Sturtevant(2011). DNA Learning Centre. Accessed October 3, 2013.

Maxam, A.M. and Gilbert, WA New Method for Sequencing DNA. (1977). P.N.A.S. Accessed October 3, 2013.

Nature Education. The Order of nucleotides in a Gene is Determined by DNA Sequencing. (2013). Accessed October 3, 2013.

Pettersson, E., Lundeberg, J, Ahmadian, A. Generations of Sequencing Technologies(2009). Genomics. Accessed October 3, 2013.

Raven, P.  FISHBiology. McGraw Hill. Accessed October 3, 2013.

Obenrader, S. The Sanger Technique(2003). Davidson College, North Carolina. Accessed October 3, 2013.

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