DNA sequencing

Chain termination sequencing involves the synthesis of new strands of DNA complementary to a single-stranded template (step I). The template DNA is supplied with a mixture of all four deoxynucleotides, four dideoxynucleotides (each labeled with a different colored fluorescent tag), and DNA polymerase (step II). Because all four deoxynucleotides are present, chain elongation proceeds until, by chance, DNA polymerase inserts a dideoxynucleotide. The result is a new set of DNA chains, all of different lengths (step III). The fragments are then separated by size using gel electrophoresis (step IV). As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded. The DNA sequence is then reconstructed from the pattern of colors representing each nucleotide sequence (step V).

Determining the exact order of the base pairs in a segment of DNA. In 1977, 24 years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method and the chemical degradation method. Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using a technique called polyacrylamide gel electrophoresis.

The DNA to be sequenced, called the template DNA, is first prepared as a single-stranded DNA. Next, a short oligonucleotide is annealed, or joined, to the same position on each template strand. The oligonucleotide acts as a primer for the synthesis of a new DNA strand that will be complementary to the template DNA. This technique requires that four nucleotide-specific reactions – one each for G (guanine), A (adenine), C (cytosine), and T (thymine) – be performed on four identical samples of DNA. The four sequencing reactions require the addition of all the components necessary to synthesize and label new DNA, including:

A DNA template
A primer tagged with a mildly radioactive molecule or a light-emitting chemical
DNA polymerase, an enzyme that drives the synthesis of DNA
Four deoxynucleotides (G, A, C, and T)
One dideoxynucleotide, either ddG, ddA, ddC, or ddT

After the first deoxynucleotide is added to the growing complementary sequence, DNA polymerase moves along the template and continues to add base after base. The strand synthesis reaction continues until a dideoxynucleotide is added, blocking further elongation. This is because dideoxynucleotides are missing a special group of molecules, called a 3'-hydroxyl group, needed to form a connection with the next nucleotide. Only a small amount of a dideoxynucleotide is added to each reaction, allowing different reactions to proceed for various lengths of time until by chance, DNA polymerase inserts a dideoxynucleotide, terminating the reaction. Therefore, the result is a set of new chains, all of different lengths.

To read the newly generated sequence, the four reactions are run side-by-side on a polyacrylamide sequencing gel. The family of molecules generated in the presence of ddATP is loaded into one lane of the gel, and the other three families, generated with ddCTP, ddGTP, and ddTTP, are loaded into three adjacent lanes. After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel.

Variations of this method have been developed for automated sequencing machines. In one method, called cycle sequencing, the dideoxynucleotides, not the primers, are tagged with different colored fluorescent dyes; thus, all four reactions occur in the same tube and are separated in the same lane on the gel. As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded, and the sequence is reconstructed from the pattern of colors representing each nucleotide in the sequence.

Researchers can use DNA sequencing to search for genetic variations and/or mutations that may play a role in the development or progression of a disease. The disease-causing change may be as small as the substitution, deletion, or addition of a single base pair or as large as a deletion of thousands of bases.

Related category

• GENETICS AND HEREDITY

Source: National Human Genome Research Institute

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