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

2. Reading the DNA sequence from an automatic sequencer
Refer to Figure 10-18, Griffiths et al., 2015.
Oligonucleotide primers can be tagged with fluorescent dyes instead of radioactive labels.
A different colored dye can be used for each of the four reactions.

3. In Search of Potential Genes
Open reading frames (ORFs) are long stretches of DNA that start with ATG and end with a stop codon. A double-stranded DNA molecule has 6 possible reading frames, 3 for each strand.

4. 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., a1-antitrypsin deficiency)
PCR Tests:
Primers can be designed to hybridize with only wild-type allele sequence such that mutant allele will not be amplified.

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

6. 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

8. 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

9. 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.

10. Gel stained with ethidium bromide.
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.
Southern blotting
Gel stained with ethidium bromide.
Autoradiogram

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

12. ~ 25,000 genes in humans are potential candidates for gene therapy.

13. Molecular Genetic Diagnostics: Sickle Cell Anemia
Electron Micrograph
HbA codes for normal b-globin and produces normal hemoglobin.
HbS produces sickled red blood cells.
Homozygotes for HbS are anemic.
HbS produces b-globin that differs from normal protein by one amino acid.

14. Molecular Genetic Diagnostics: Sickle Cell Anemia

15. Molecular Genetic Diagnostics: Sickle Cell Anemia

16. Methods of introducing a transgene
Solution of DNA to be studied is injected into host cell.
Recombinant DNA can be delivered into host cell using a viral vector.
Refer to Figure 10-23, Griffiths et al., 2015.

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