Ch 10: part 2 Lecture Notes Flashcards
What is cloning (vs. organism cloning?)
Cloning means you will make lots of copies of a DNA sequence. Organism cloning is like Dolly the Sheep where you make a copy of a specific organism.
What was one of the earlier successes of DNA cloning?
Insulin production in bacteria. Human genes inserted into a plasmid
What are the basic steps to DNA cloning?
1) Take the DNA out of the cell (DNA preparation)
2) Cut the sequence out (digestion with restriction enzymes)
3) Glue multiple pieces back together (ligation with ligase)
4) Make more copies of the DNA (PCR)
5) Put the DNA back into the cell (Transformation)
What is a plasmid?
A small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small, circular, double-stranded DNA molecules in bacteria.
Makes a good cloning vector
What makes a good cloning vector?
A plasmid
Why do bacteria have plasmids?
There is an advantage to being able to transfer beneficial genes (like abx resistance) to bacteria that are close kin. This is called horizontal gene transfer.
Horizontal gene transfer
When bacteria can pick up DNA from the environment, allowing them to share genes (hopefully beneficial) with other nearby bacteria.
What are the important features on a plasmid?
1) selection marker (ie antibiotic resistance gene)
2) origin of replication
3) promoter
4) ribosome binding site or Kozak
5) transcription termination sequence
What is the selection marker on a plasmid?
Something that gives the bacteria an advantage, and a reason to have the plasmid stick around and continue making copies. ie abx resistance. Copying plasmids takes a lot of energy, so it only makes sense to do so when it helps the cell.
How does a PCR work?
It amplifies DNA
1) design a DNA to use as a primer that bind to the sites you want.
2) heat up to separate the strands
3) cool down, let the primer bind, and let the replication happen. Some of the initial fragments will be too long, but as you copy, the correct sized ones will increase exponentially.
4) repeat over and over
30 cycles takes about one hour, so the device needs to cool down and heat up quickly and precisely.
DNA restriction enzymes
cut the DNA as a specific spot (a palindromic sequence). Leave a sticky overhang to which other things can bind.
Why are there restriction enzymes?
It’s a bit like CRISPR, it cuts foreign material DNA that invades the cells.
Why do you need two cuts with the restriction enzymes?
Create two different sticky ends, so you can correctly orient the gene you want inserted. There are two restriction enzymes as well. If there were only one, the over hangs would match, and could stick back together easily.
How do you tell whether the cuts with the restriction enzyme was successful?
You should have two shorter fragments, and can run a gel to check the lengths
Ethinium bromide
A molecule that sticks in between double-stranded DNA and lights up. Can be used as a marker for gel electrophoresis.
How do you select for the bacteria with the plasmid of interest?
Likely only a small percentage of the bacteria will pick up the plasmid you want. The plasmid needs to also contain a selection marker (like antibiotic resistance), so that the cells without it can be eliminated.
Can the e coli replicate this plasmid?
will e coli cells carrying this plasmid produce functional amipcillin resistance protein?
Will e coli cells carrying this plasmid produce functional GFP?
Will transforming this plasmid into ampicillin-sensitive e coli cells result in bacteria that are ampicillin resistance and glow in UV light?
Can the e coli replicate this plasmid? Yes (it has a replication origin)
will e coli cells carrying this plasmid produce functional amipcillin resistance protein? (yes, it has the right promoter in front of the abx resistance gene)
Will e coli cells carrying this plasmid produce functional GFP? (no, it has a jellyfish promoter)
Will transforming this plasmid into ampicillin-sensitive e coli cells result in bacteria that are ampicillin resistance and glow in UV light? No, it will and resistant, but won’t glow.
Note: sometimes a jellyfish promoter will work in an ecoli, but usually something form another species implies that it won’t work.
What are the basic principles of genome editing?
How does gene disruption technology work in eukaryotes?
The exact numbers don’t matter, but the longer a genome, the longer a sequence needs to be in order for it to be specific to one spot.
How does the zinc finger work to edit genomes?
Each zinc finger recognizes 3 base pairs, and it likes to start with a G, so not all sequences are possible
The zinc finger does not cut DNA on its own, it needs a nuclease to be able to cut.
The nuclease needs to be on either side of the strand to cut the DNA (it is a heterodimer), so for one cut site, you need two proteins.
How does CRISPR work?
There is a guide RNA:
part binds to CRISPR and is always the same
part is the target site for where the cut should be.
The target site needs to be followed by NGG
Explain how to do a genome editing project
