Genomics and Biotechnology

Transcription

1 Genomics and Biotechnology

2 Expansion of the Central Dogma DNA-Directed-DNA-Polymerase RNA-Directed- DNA-Polymerase DNA-Directed-RNA-Polymerase RNA-Directed-RNA-Polymerase RETROVIRUSES

3 Cell Free Protein Synthesis

4 Mutations and Polymorphisms

5 Mutations and Polymorphisms An error in base sequence that is carried along during DNA replication is called a mutation. Mutation commonly refers to variations in DNA sequence found in a very small number of individuals of a species. Some mutations result from spontaneous and random events. Others are induced by exposure to a mutagen an external agent that can cause a mutation. Viruses, chemicals, and ionizing radiation can all be mutagenic.

6 Mutations and Polymorphisms Polymorphisms are variations in the nucleotide sequence of DNA that are common within a given population. Most polymorphisms are simply differences in the DNA sequence between individuals due to geographical and ethnic differences and are part of the biodiversity exhibited by life on earth. The vast majority of polymorphisms seen have neither advantageous nor deleterious effects, some have been shown to give rise to various disease states.

7 Mutations and Polymorphisms The replacement of one nucleotide by another in the same location along the DNA sequence is a single-nucleotide polymorphism. The biological effects of SNPs range from negligible to normal variations such as those in eye or hair color, to genetic diseases. In addition to producing a change in the identity of an amino acid, a SNP might specify the same amino acid, or it might terminate protein synthesis by introducing a stop codon.

8 The sickle cell mutation causes hemoglobin molecules to clump together in an abnormal manner. The valine in position 6 adheres to a notch on the opposite side of another molecule of Hb, causing long chains to form.

9 In sickle cell anemia, the normal hemoglobin molecule mutates by exchanging the 6th amino acid on the beta chain from glutamic acid to valine. Normal Hb has the genotype SS. Sickle cell anemia occurs when an individual inherits two recessive alleles (ss). Sickle cell trait exists when one inherits the heterozygous condition (Ss).

10 Sickle Cell Anemia

11 Sickle cell anemia occurs when an individual inherits two recessive alleles (ss). Sickle cell trait exists when one inherits the heterozygous condition (Ss). Curiously, the malaria parasite (Plasmodium falciparum) does not survive in these individuals; they may have a slight anemia, but they survive better than either normal individuals (SS- who often die of malaria), or those who die of sickle cell disease (ss).

12 Sickle Cell Anemia Comparison of the distribution of malaria (left) and sickle-cell anaemia (right) in Africa

13 The sickle cell mutation causes hemoglobin molecules to clump together in an abnormal manner. The valine in position 6 adheres to a notch on the opposite side of another molecule of Hb, causing long chains to form.

14 The Genetic Code Glu Val

15 Insulin Gene Mutations

16 The Genetic Code Cys Gly

17 Restriction Endonucleases

18 Restriction Endonucleases Clues from Bacteriophages

19 Restriction Endonucleases - Specificity

20 For their 1970 discovery of restriction endonucleases (often called by the shorter name restriction enzymes) Werner Arber, Hamilton Smith, and Daniel Nathans received the 1978 Nobel Prize for Physiology or Medicine. HindII was the first restriction enzyme to be isolated, but many others were later discovered and characterized. Restriction enzymes are produced by bacteria, in which they function as a simple immune system A restriction enzyme protects the bacterium producing it from viral infection by chopping up invading viruses, which are composed of either RNA or DNA. Each restriction enzyme recognizes and cuts a specific, short nucleotide base sequence, not found in the genome of the bacterium that produces it. For example, the restriction enzyme EcoRI will bind a DNA helix (right) and cut it at (and only at) points where the nucleotide sequence GAATTC occurs (see lower figure at right). This specific 6- nucleotide sequence is the restriction site of EcoRI (other restriction enzymes have other restriction sites). The green line in the figure indicates the path of the cut created by the enzyme. But restriction enzymes will cut not only the DNA and RNA of viruses invading a bacterium, but also DNA and RNA of any kind. This makes them incredibly useful for constructing composite DNA sequences and for carrying out molecular genetic investigations.

21 Restriction Endonucleases - How Bacteria Protect Themselves

22 Recombinant DNA Production A restriction enzyme cuts DNA from two different organism at the same locus.

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24 Recombinant DNA Production It is possible to cut a gene out of one organism and splice it into the DNA of a second organism. Bacteria provide excellent hosts for recombinant DNA. Bacterial cells contain part of their DNA in small circular pieces called plasmids, each of which carries just a few genes.

25 Construction of a Recombinant Plasmid Recombinant DNA is produced by cutting the two DNA segments to be combined with the same restriction endonuclease. The result is DNA fragments with complementary sticky ends. The two are mixed in the presence of a DNA ligase enzyme that joins them together by re-forming their phosphodiester bonds, reconstituting the now-altered plasmid.

26 Construction of a Recombinant Plasmid

27 Recombinant DNA Production The altered plasmid is inserted back into a bacterial cell where the normal processes of transcription and translation synthesize the protein encoded by the inserted gene. Bacteria multiply rapidly; there are soon a large number of them, all containing the recombinant DNA and all manufacturing the protein encoded by the recombinant DNA. Huge numbers of the bacteria can be put to work as a protein factory. Proteins manufactured in this manner have already reached the marketplace, including human insulin, human growth hormone, and blood clotting factors for hemophiliacs.

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30 Plasmid Insertion