Week 10 Gene Isolation and Manipulation

Southern, Northern, Western

It separates mixtures of DNA fragments, RNAs or proteins based on physical properties such as size and charge Gel is the matrix used t separate molecules and electrophoresis is the voltage that is applied DNA and RNA have overall negative charges so will migrate from negative to positive depending on their size

Transfer to a membrane Addition of the probe

Radioactive single stranded nucleic acids that are complementary to the nucleic strand of interest - will hybridize to the complementary strand -unbound probe is washed away and hybridization is revealed by autoradiography (x-ray)

Palindromic

Since it is do big, it is easier to work with and be analyzed in smaller fragments

Restriction enzymes

Quantifying PCR, measures the amount of DNA product in “real time” during each PCR cycle. -measures the intensity of a fluorescent signal

a method for isolating a particular sequence of DNA from a complex mixture of different DNA sequences - needs to be inserted into a vector

Solves problem where the gene could orient itself either way into the vector/the vector could just reanneal - force directional cloning uses two different restriction enzymes to make sticky ends not complementary to each other, will have compatible ends on only one side each, when they come together and when they anneal they can only go one way Also means that the plasmid can’t just reanneal to itself

Polylinkers Origin of Replication Drug Resistance Gene

Gene libraries represent all of the genes in an organisms whereas cDNA libraries represent only those genes that were expressed in the cell that were a source of mRNA

Colonies on a petri dish are transferred to a membrane Lyse bacterial cells and denature DNA Add probe Autoradiograph the locate the desired clone Pick up positive clone -Grow and amplify the colony

Use PCR primers with restriction sites at their 5′ end to produce PCR products with sticky ends Can also use Gibson Assembly (multiple linear DNA fragments that have sequence similarity at their ends)

Dideoxy sequencing/Sanger sequencing

-Chemical: lipid vesicle being engulphed Physical: electroporation, biolistic particle delivery, microinjection Biological: bacteria or viruses

Transgenic yeast cells are generated by homologous recombination between a yeast chromosome and a plasmid that is transformed into yeast and carries a gene of interest

Transgenic plants are generated by random insertion into a chromosome of a Ti plasmid that carried a gene of interest and is delivered by Agrobacterium tumefaciens Ti plasmid causes an infected plant to produce uncontrolled growths called tumors or galls -Scientists are able to eliminate all of the T-DNA sequence between the borders (including the tumour causing gene) and replace it with a gene of interest and a selectable marker

Transgenic worms are generated by injection of a plasmid containing a gene of interest into the gonad.

An inactive gene is targeted to replace the functioning gene in a culture of ES cells (embryonic stem cells)

An inactive gene is targeted to replace the functioning gene in a culture of ES cells, producing ES cells containing a gene knockout ES cells containing the inactive gene are transferred to mice embryos (Targeted insertion of transgene by homologous recombination) Knockout mice are identified and bred to produce mice of a known genotype (selection of cells with gene knockout)