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. Restriction Mapping
DNA is restriction digested with restriction enzymes, individually (single-enzyme digest) and in combination (double digest).
The restriction fragments are subjected to electrophoresis.
The fragments are identified, either using UV absorbing dye or labeled probe.
Double digest determines if fragment produced by one enzyme contains restriction sites for the other enzyme.
Fragments are aligned by size.
Enzyme 1: 8 kb, 6 kb, 3 kb or 3 kb, 6 kb, 8 kb
6 kb, 8 kb, 3 kb or 3 kb, 8 kb, 6 kb
8 kb, 3 kb, 6 kb or 6 kb, 3 kb, 8 kb
Enzyme 2: 10 kb, 7 kb or 7 kb, 10 kb
Double Digest: 3 kb fragment is split into
2 kb and 1 kb fragments.

4. Restriction Fragment Length Polymorphism (RFLP)
Individuals can be identified according to RFLP genotype. RFLP locus could be linked to a gene, and thus be used as a diagnostic marker.

5. Use of Restriction Fragment Length Polymorphism
I. Marker locus
II. Diagnostic
A. Medicine
B. Forensics
III. Assessment of Genetic Variation
A. Within and between populations
B. Within and between species

6. Restriction Mapping versus RFLP Mapping
I. Restriction Mapping
A. Based on physical analysis of DNA
B. Based on restriction sites with no variation
C. Mostly short-range (fine-scale) maps
II. RFLP Mapping
A. Based on recombination analysis of matings
B. Based on restriction-site variation between homologous chromosomes
C. Mostly longe-range (coarse-scale) maps

7. 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

8. 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’

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

10. 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

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

12. 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.

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

14. Reading the DNA sequence from an automatic sequencer
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.

15. 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.