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.