Week 10 Gene Isolation and Manipulation Flashcards
What types of blotting is used for DNA, RNA, and proteins respectively
Southern, Northern, Western
What does gel eletrophoresis do?
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
What are the steps that follow gel electrophoresis?
Transfer to a membrane
Addition of the probe
What are the probes in Southern and Northern blotting?
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)
Restriction enzymes are ___ on opposite strands
Palindromic
Why does cutting chromosome sized DNA occur
Since it is do big, it is easier to work with and be analyzed in smaller fragments
____ cut DNA at specific
sequences, producing
fragments with staggered
or blunt ends.
Restriction enzymes
What is qPCR?
Quantifying PCR, measures the amount of DNA product in “real
time” during each PCR cycle.
-measures the intensity of a fluorescent signal
DNA cloning
a method for isolating a particular sequence of DNA from a complex mixture of different DNA sequences - needs to be inserted into a vector
Force directional cloning
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
Practical features of plasmid vectors
Polylinkers
Origin of Replication
Drug Resistance Gene
Genomic library vs cDNA library
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
How to identify a clone of interest from a genomic of cDNA library
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
Cloning by PCR
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)
What is the most common way to sequence DNA?
Dideoxy sequencing/Sanger sequencing
Transgenes are introduced into eukaryotic cells by chemical, physical, and biological methods such as:
-Chemical: lipid vesicle being engulphed
Physical: electroporation, biolistic particle delivery, microinjection
Biological: bacteria or viruses
Genetic engineering in yeast
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
Genetic engineering in plants
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
Transgenesis in C. elegans
Transgenic worms are generated by injection of a plasmid
containing a gene of interest into the gonad.
Gene knockout
An inactive gene is targeted to replace the functioning gene in a
culture of ES cells (embryonic stem cells)
The three stages of gene knockout
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)
