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DNA Sequencing

DNA Sequencing. DNA sequencing is employed to determine the ordered DNA nucleotide sequence of double-stranded DNA. Dideoxynucleotide DNA Sequencing. Employs DNA synthesis in the presence of small amounts of nucleotides that contain the sugar dideoxyribose instead of deoxyribose.

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DNA Sequencing

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  1. DNA Sequencing

  2. DNA sequencing is employed to determine the ordered DNA nucleotide sequence of double-stranded DNA.

  3. Dideoxynucleotide DNA Sequencing • Employs DNA synthesis in the presence of small amounts of nucleotides that contain the sugar dideoxyribose instead of deoxyribose. • Dideoxyribose lacks the 3’ OH group, which is essential for attachment of the next nucleotide in a growing DNA strand. • Incorporation of a dideoxynucleotide terminates further elongation of a DNA strand.

  4. Structures of deoxyribose and dideoxyribose sugars.

  5. Requirements of dideoxy sequencing. • Four DNA synthesis reactions are carried out. • Each reaction contains many copies of the single-stranded DNA template to be sequenced, a labeled single-stranded DNA oligonucleotide primer, all four deoxynucleotides and a small amount of one of the four dideoxynucleotides and DNA polymerase.

  6. Dideoxy method of DNA sequencing.

  7. Automated DNA sequencing. • The dideoxynucleotides that terminate synthesis are each labeled with a different fluorescent dye (G, black; A, green; T, red; C, blue. • Because the colors distinguish the products of DNA synthesis that terminate with each dideoxynucleotide, the products of all the synthesis reactions can be mixed together in the same tube and separated by electrophoresis in a single lane. • A laser scans the gel and the color of the fluorescence for each DNA fragment is recorded in a computer.

  8. Automated DNA sequencing.

  9. Shotgun DNA Sequencing. • Invented by Craig Venter. • Take chromosomal DNA, cut it up into many small fragments using several different restriction enzymes. • Clone the fragments in circular plasmids that can replicate in bacteria. • Sequence the small fragments that were cloned in bacteria plasmids via automated dideoxy sequencing. • Computer analysis is used to recognize DNA fragments that overlap. A computer program then joins sequences into a continuous DNA sequence.

  10. Shotgun sequencing strategy.

  11. Shotgun sequencing was first done on bacterial genomes. Then it was done on eukaryotic genomes.

  12. Clinical use of dideoxynucleoside analogs. • A nucleoside is a base attached to a sugar without a phosphate group. • A nucleoside analog is a molecule similar, but not identical in structure, to a nucleoside. • The nucleoside, rather than the nucleotide is used in therapy because the nucleotide, having a highly negative charged phosphate group, cannot cross the cell membrane easily.

  13. Some drugs used in the treatment of AIDS.

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