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

2. Refer to Figure 7-26 from Introduction to Genetic Analysis, Griffiths et al., 2015.

3. The Problem of Replicating Chromosome Ends
telomere 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ A B A B A B
RNA primer
removal and
DNA ligation + +
DNA
Replication 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
Last section of lagging strand cannot be primed. Result is a shorter chromosome after each round of replication.

4. The Problem of Replicating Chromosome Ends
Here is another angle. Note again that the last section of lagging strand cannot be primed. A shorter chromosome is produced after each round of replication.
It is theorized that as cells age (generations), telomeres shorten, eventually leading to their death.
Is there a mechanism to maintain chromsome length?

5. Telomere Lengthening
Telomerase (a reverse transcriptase) adds repeats to telomeric DNA.
It carries an RNA molecule that serves as template for DNA synthesis.
Refer to Figure 7-27 from Introduction to Genetic Analysis, Griffiths et al., 2015.

6. Telomere Lengthening
Refer to Figure 7-27 from Introduction to Genetic Analysis, Griffiths et al., 2015.

7. The Telomeric Cap Structure
Telomeric end is protected by a “cap”.
It consists of TRF1 and TRF2 (that bind to telomeric repeats) and proteins such as WRN* that bind to TRF1** and TRF2**.
* Werner Syndrome protein
** TTAGGG Repeat Binding Factor
Refer to Figure 7-28 from Introduction to Genetic Analysis, Griffiths et al., 2015.

9. 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

10. Making Recombinant DNA: Donor DNA
Genomic DNA:
DNA obtained from chromosomes of an organism
Complementary DNA (cDNA):
double-stranded DNA version of mRNA obtained by reverse transcription
Chemically Synthesized DNA:
DNA sequence obtained by automated chemical reactions

11. Cutting DNA: Restriction Endonucleases

12. Formation of a recombinant DNA molecule
Circular ds DNA is cut with one restriction enzyme.
Both restriction fragments are linear and have sticky ends (in this case).
Linear ds DNA is cut with the same restriction enzyme.
By complementary base pairing, the sticky ends can hybridize.
The result is a recombinant DNA molecule.
Refer to Figure 10-2 from Introduction to Genetic Analysis, Griffiths et al., 2015.

13. Inserting a gene into a recombinant DNA plasmid
Vector is a cloning vehicle.
Both vector and donor DNA are cut with the same restriction enzyme.
Restriction fragments are mixed; sticky ends hybridize.
Recombinant vector is the result.
DNA ligase seals gaps by forming phophodiester linkages.
Refer to Figure 10-5 from Introduction to Genetic Analysis, Griffiths et al., 2015.

14. How amplification works
Recombinant vectors are introduced into bacterial host cells.
Replication and cell division produce many copies of the recombinant vector.
Clones of donor DNA fragments result.
Refer to Figure 10-8 from Introduction to Genetic Analysis, Griffiths et al., 2015.

15. Choice of Cloning Vectors: Criteria
Small Size:
Convenience of manipulation
Capability of Prolific Replication:
Ease of amplification of donor DNA fragment
Convenient Restriction Sites:
Single location for insertion of donor DNA
Ease of Identification:
Quick recovery of recombinant DNA

16. Examples of Cloning Vectors
Bacterial Plasmids:
* Circular double-stranded DNA
* Replicates independently of chromosomal DNA
* Selectable markers for transformation
Bacteriophages:
* Phage l - clone DNA up to 15 kb

17. Vectors for Larger DNA Inserts
Fosmids:
Hybrid between l phage DNA and plasmid DNA - can carry inserts 35-kb to 45-kb
PAC:
P1 Artificial Chromosome (derivative of bacteriophage P1) - can carry inserts 80-kb to 100-kb
BAC:
Bacterial Artificial Chromosome (derivative of F plasmid) - can carry inserts 150-kb to 300 kb
YAC:
Yeast Artificial Chromosome - can carry inserts larger than 300-kb

18. Modes of delivering recombinant DNA into bacterial cells
Refer to Figure from Introduction to Genetic Analysis, Griffiths et al., 2015.
(a) Plasmid DNA is introduced into host cell by transformation.
(b) Fosmids are introduced in phage heads by transduction. Once inside, they replicate as large plasmids.
(c) Phage vectors are introduced by infection.