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. Other Useful Approaches
1. Single Nucleotide Polymorphisms (SNPs)
Individuals differ in single nucleotides (every 11 to 300 bp in interval).
Simple-Sequence Length Polymorphisms (SSLPs)
Very short repetitive DNA sequences are more polymorphic than RFLP sequences. These are also called Variable Number Tandem Repeats (VNTRs)
- Minisatellite Markers
- Microsatellite Markers

3. Simple-Sequence Length Polymorphisms
1. Minisatellite DNA
These are 1 to 5 kb in length consisting of repeats 15 to 100 nucleotides in length and are identified by Southern analysis.
Microsatellite DNA
These are tandem repeats of dinucleotides, commonly stretches of CA.
These are identified by gel electrophoresis of PCR products.
5’ C A C A C A C A C A C A C A 3’
3’ G T G T G T G T G T G T G T 5’

4. Refer to Figure 4-15, Griffiths et al., 2015.

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. Dideoxy DNA Sequencing
Chain-terminating (dideoxy) nucleotide
Use of dideoxy nucleotide in primer extension reaction will randomly arrest DNA synthesis.

7. Dideoxy DNA Sequencing
4 different reactions are conducted, each with a different type of dideoxy nucleotide.
Fragments are separated by electrophoresis.
Banding pattern is used to infer the base sequence of the original template strand.
Refer to Figure 10-17, Griffiths et al., 2015.

8. This is base sequence of synthesized strand.
Migration
Sequencing Gel Using
Radioactive
Primer
Remember…
This is base sequence of synthesized strand. 3’ 5’

9. 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.

10. 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.

11. 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.

12. 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

13. 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

15. 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

16. 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.

17. 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