Biochemical methods

Enzyme activity determination allows detecting metabolic disorders (e.g. hereditary disease phenylketonuria).

Studying of subcellular fractions chemical composition by chromatography, gel electrophoresis, qualitative reactions show proteins and amino acids composition.

Autoradiography, or tracer method, based on the radioactive isotopes introduction into the cells: tritium ³Н, carbon ¹⁴С, sulfur ³⁵S, phosphorus ³²Р, iodine ¹³¹I and others. In a certain intervals from the treated material samples are prepared. Then the surface of these samples is blanketing with thin layer of photo emulsion with silver halide. If there is radioactive isotope in a certain parts of the cell glow appears. Glow localization indicates areas of the cells with intensive biochemical reactions. For example, with tritium in the medium it is possible to visualize DNA replication.

Dideoxy method of DNA sequencing (or Sanger method) developed by Fred Sanger. This method acts by interrupting the ability of the polymerase to add new nucleotides at specific points along the DNA chain.

Chemically modified nucleotide, called a dideoxynucleotide (ddNTP), replaces one of the nucleotides in the chemical mixture. A ddNTP is missing the 3′-hydroxyl group. Because of this, the DNA polymerase is unable to synthesize a phosphodiester bond between a dideoxynucleotide and the next nucleotide in the chain. This ceases DNA replication at this point. So, this method is sometimes referred to as chain termination.

A researcher prepares four identical reaction tubes. Each tube contains a single stranded copy of the DNA for sequencing and a primer for replication reaction beginning. In each tube the researcher places normal A, G, T, and C nucleotides, and a small quantity of dideoxy A (ddATP) in the first tube, dideoxy G (ddGTP) in the second tube, dideoxy T (ddTTP) in the third tube, dideoxy G (ddCTP) in the fourth tube. Once the polymerase begins replication, it will occasionally incorporate one of the ddNTPs into the strand, thus stopping replication. In the tube containing ddATP there will be an assortment of DNA fragments, varying in length, each with a ddATP at the end of the fragment.

The four reactions are then loaded into a polyacrylamide gel and exposed to an electrical current to separate the fragments by size. Smaller fragments will migrate further in the gel than large fragments. In order to detect the results of the reaction, the dideoxynucleotides are labeled with radioactive phosphorous (³²P). The gel is then placed with a piece of photographic film and developed after 24 hours of exposure.

The Results of a Dideoxy Sequencing Reaction

(Courtesy of Ricochet Productions)

To read the results, start with the band at the very bottom of the gel. This represents the smallest fragment. Since it is in the C column, the reaction must have been terminated with the incorporation of a dideoxy C nucleotide into the strand at that point. The next smallest fragment is located under the T column, indicating that the next nucleotide in the sequence is a thymine. The results are read in a ladder-like configuration up the gel. The sequence indicated is CTTCAGGTACAGTAA.