Genomic libraries Flashcards
How do we define genome?
A complete set of DNA of an organism
How to determine the size of a genomic library (number of unique clones)?
N = ln(1-P) / ln(1-f)
P - probability that sequence is present in at least one clone
f - fractional size = clone insert size/ genome size
What is ‘partial digestion’ and what does it aim to achieve?
It is a further digestion with a different restriction enzyme, whose avg distance between restriction sites is smaller than the desired DNA fragment length. It aims to achieve a uniform length of the DNA library inserts.
What is the choice of appropriate vector based on?
On the optimal insert capacity matching with the avg length of DNA fragments of the cut genome
What gene trasfer mechanism is used to introduce Cosmid DNA to the bacterium?
Transduction via phage
What gene trasfer mechanism is used to introduce BAC DNA to the bacterium?
Transformation from the surroundings
What gene trasfer mechanism is used to introduce YAC DNA to the bacterium?
Electroporation - bombardment of cellular membrane with high conductivity particles coated with DNA
What is a stable manipulation?
A genetic modification that is passed to the progeny
What is the difference between manipulating the genomes of cell lines and animals?
In animals the mutation needs to go through the germ line, otherwise it will not be present in all of the organisms cells.
What is addition by random integration?
An early genome manipulation technique, involving transporting the labelled DNA fragments into the cells with different mechanism and using the marker to idenitify cells with succesfully integrated DNA inserts.
What is homologous recombination?
A method of genomic manipulation involving recombination between similar DNA molecules.
What is site specific recombination?
A method of genomic manipulation involving using a recombinase (e.g. Cre), that catalyzes directionally sensitive DNA exchange reactions between short (30–40 nucleotides) and a target site sequences (lox) that is specific to each recombinase.
What is guide RNA manipulation?
CRISPR-Cas9 system - Cas9 nuclease uses a guide RNA sequence to target a specific complementary DNA sequence for cleavage.
How are genetically modified mice produced?
1) targeting vector is synthesised and introduced to the ES cells (from mice blastocyst)
2) Targeting vector is integrated into the genome via gene targeting(insertion/deletion/replacement)/random integration (insertion) mechanism
3) ES cells carrying the modification are selected and injected to the blastocyst
4) Blastocysts are injected into mother, which give birth to chimeric mice (parts of the body express the mutation, because their cells originte from the modified cells)
5) Breed chimeric mice until a fully mutated mice is achieved.
