1. Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

2. Molecular Genetic Diagnostics Restriction Site Analysis: Mutant allele and wild-type allele could differ in presence or absence of restriction site. (e.g., sickle cell anemia) Probe Hybridization: Synthetic probe could distinguish between mutant and wild-type alleles by hybridization at elevated temperatures. (e.g.,  1 -antitrypsin deficiency) PCR Tests: Primers can be designed to hybridize with only wild- type allele sequence such that mutant allele will not be amplified.

3. Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

4. Detection and Isolation of Target Molecules For Nucleic Acids 1. cDNA of a specifc gene 2. Homologous DNA of gene from related organism 3.Synthetic DNA based on amino acid sequence of protein For Proteins 1.Antibodies

6. Separation of Molecules by Gel Electrophoresis Subject molecules to electrical field in a matrix. Separation of molecules is based on: 1. net charge 2. size 3.shape

7. Blotting Techniques Southern Analysis DNA fragments are electrophoresed and probed with DNA or cDNA for specific sequence. Northern Analysis RNA molecules are electrophoresed and probed with cDNA for specific sequence. Western Analysis Proteins are electrophoresed and probed with antibody for specific protein.

8. Southern Analysis 1. Fragmented DNA molecules are separated by gel electrophoresis. 2. After electrophoresis, gel is placed in container of buffer and nitrocellulose filter is placed on top of the gel to transfer DNA by capillary action to the filter. 3. Filter is incubated with labeled single-stranded probe (often radioactive probe is used). 4. After unbound probe is removed, the filter is placed on an X-ray film for autoradiography. 5. Probe hybridizes only with complementary fragments. Gel stained with ethidium bromide. Autoradiogram Southern blotting

9. Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

10. ~ 25,000 genes in humans are potential candidates for gene therapy.

11. Molecular Genetic Diagnostics: Sickle Cell Anemia Electron Micrograph Hb A codes for normal  -globin and produces normal hemoglobin. Hb S produces sickled red blood cells. Homozygotes for Hb S are anemic. Hb S produces  -globin that differs from normal protein by one amino acid.

12. Molecular Genetic Diagnostics: Sickle Cell Anemia

14. Solution of DNA to be studied is injected into host cell. Recombinant DNA can be delivered into host cell using a viral vector. Methods of introducing a transgene

15. Bombardment with DNA-coated tungsten particles introduces DNA into host cell. Under favorable conditions, DNA can be taken up by host cell by transformation. Methods of introducing a transgene

16. Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation

17. 1.Introduction of foreign genetic material into a host organism. 2. Ability of the new genetic material to be transmitted to offspring of the host. 3. Potential of the new genetic material to be expressed in the host. 4. Transgenic animals or transgenic plants are the result of this process. Transgenesis