What are the 2 approaches to genome sequencing?
Hierarchical and Shotgun
Construction of linkage map vs fragmentation of whole genome into plasmids
What is Next Generation sequencing?
Immobilizing and amplifying a template strand to pruduce identical fragments
High throughput technology
Give 4 things you can do with a genome sequence
Store it, Identify all the genes, Compare with other species, Test gene functions
What percentage of the human genome are introns and regulatory sequences?
Give 5 practical benefits of genome projects
Molecular medicine, Energy sources, Bioarchaeology, Forensics, Agriculture
Why do we clone cDNA copies of genes?
Only genes are cloned, Removes introns, mRNA can't be cloned, keep a physical record of protein coding sequences
Give 3 methods of screening cDNA libraries
Hybridization, Immunodetection, Complementation
Labelled probes, Antibodies, Mutants
Give the 2 types of probe used in hybridization
Homology or degenerate
Carries DNA of a related organism, Carries a degenerated sequence inferred from a known protein sequence
What 2 types of labels are used in hybridization?
Radioactive(32P or 33P) or non-radioactive (nucleotides coupled with DIG)
Give 2 advantages of radioactive labels
Robust, Easy to detect
However, they are hazardous, complicated, short-lived
Give 2 enzymes that assist in incorporating labels into DNA
Terminal transferase, Taq polymerase
Give 3 things that can act as the required antigen in immunodetection
Whole protein, Close homologue, Synthetic peptide
Why do we infect bacteria using cDNA-containing bacteriophages during immunodetection?
They replicate in the bacteria and produce the required protein, then destroy the bacteria to release the protein
What is the principle of complementation screening?
Use the activity of the protein to rescue a mutant, it will be easily detected amongst other clones
Powerful, easy, fast and cheap but not always applicable
Give 3 considerations when selecting an expression system
Size of target protein, solubility of target protein and Post-translational modifications
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