BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

DNA Sequencing Methods

The process of determining The nucleotide sequence of a DNA fragment is called sequencing.

The most common sequencing method is the dideoxynucleotide method. The name of the method derives from the term "dideoxynucleotide".

A dideoxynucleotide is a deoxynucleotide lacking the 2'- and

3'-hydroxyl groups in the ribose molecule.

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It is known that DNA Synthesis occurs through The addition of a nucleoside triphosphate to the 3'-OH group of a nucleotide. The incorporation of a dideoxynucleotide halts the elongation of the polynucleotide chain because the dideoxynucleotide lacks the 3'-OH group.

For sequencing, four DNA synthesis reactions are carried out simultaneously in different test tubes using the template whose nucleotide sequence is to be determined. Each reaction takes place in the presence of one of the four dideoxynucleotides. Incubating the single-stranded DNA designated for sequencing with a primer, DNA polymerase I, dATP, dGTP, dCTP, and dTTP ensures the copying of the DNA fragment. One of the triphosphates must be radioactive so that the synthesized DNA molecules are labeled. A unique set of oligonucleotides is synthesized in each of the test tubes.

Electrophoresis is used to separate DNA molecules: DNA molecules migrate in an electric field along a polyacrylamide gel plate at a speed dependent on chain length—the shorter the DNA chain, the faster it moves. Each nucleotide addition alters the mobility of the chain. Chains with different mobility correspond to bands identified by autoradiography.

Using specialized devices (sequencers), the DNA Sequencing process is carried out automatically.

Sequencing of Long Nucleotide Sequences

Using a primer and the dideoxy method, the first 250–300 NUCLEOTIDES are sequenced. Based on the sequencing results, a second primer is synthesized to determine The sequence of the next 250–300 nucleotides. This method is called "primer walking". The method makes it possible to sequence very long DNA fragments.

A New DNA Sequencing Method

Professor O. Aksimentiev and his colleagues have developed a device capable of recording nucleotide sequences as a DNA molecule passes through a nanopore in a silicon microchip. In an electric field, a DNA molecule is able to pass through pores 2 nm in diameter. As the DNA molecule moves through the nanopore, its electric field induces specific electrostatic potentials that can be detected and decoded.

This method allows for The Study of the mechanical properties of DNA and enables rapid and accurate genome sequencing. As is known, Human Genome sequencing has a high cost. The cost of sequencing The Human Genome using Aksimentiev's method is $1,000.