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Chapter 18 : DNA Sequencing: The Terminators
Determining the base sequence of a piece of DNA is a critical step in applications of biotechnology. In DNA replication, a nucleotide complementary to the template base is brought into position. The DNA polymerase then adds it to the growing DNA strand by forming a bond between the 5' phosphate group of the new nucleotide and the 3' OH group of the previous nucleotide. The chain terminators used in DNA sequencing are the dideoxynucleotides. The newly synthesized molecules are separated by size and visualized by exposing the gel to photographic film. The A reaction lane shows bands that correspond in length to the site of each T in the template. The G reaction lane shows bands whose lengths correspond to the site of template C’s, and so on. The sequence of the new DNA strand is “read” from the sequencing gel by starting at the bottom (the shortest new molecule) and reading upward. As in PCR, the mixture is put through temperature cycles of denaturation, hybridization, and DNA synthesis. Unlike a PCR, only one primer is present, so DNA synthesis occurs using only one strand of the parental DNA as a template. The AIDS virus encodes a special enzyme, reverse transcriptase, that synthesizes DNA using the viral RNA as a template. HIV infects cells as an RNA genome packaged with the enzymes reverse transcriptase and integrase inside a protein envelope. The herpesviruses are DNA viruses with relatively large genomes. Herpesvirus diseases can now be treated with the chain terminator acyclovir.
Key Concept Ranking
- DNA Synthesis