Lab 6: DNA cloning Flashcards
is the process by which pieces of DNA are transferred from one location (e.g. genome) to another location
DNA cloning
allowed scientists to study genes (and their regulatory sequences)
DNA cloning
what does DNA cloning allow scientist to do
a) Study the function of individual genes more efficiently
b) Amplify a given piece of DNA
c) Create new combinations of DNA
The average eukaryotic genome contains how many genes
20,000-40,000 different genes
what allows scientists to more efficiently mutate the gene, alter its expression levels, and ultimately dissect its function.
Being able to selectively remove a gene of interest from the genome and move it away from all of the other genes
amplifies DNA using purified enzymes in a plastic tube
PCR
amplifies DNA by moving a piece of DNA into a cloning vector (plasmid) and introducing that vector into a bacterial cell.
DNA cloning
what happens everytime bacteria divides
the vector is copied via normal cellular DNA replication.
re-extracted from the bacteria and the fragment is cut back out
amplified vectors
DNA amplification utilizes the power of
bacterial cell division
utilized DNA cloning technology to create
a new cancer therapy
how did they utilized DNA cloning technology to create a new cancer therapy
I removed a cell death gene (which is normally only expressed when a cell wants to kill itself) from the human genome and put it into a plasmid that contains a constitutively active promoter. Thus, this gene will now be expressed at much higher rates than its normal counterpart. As a result, when introduced into a cancer cell, this gene will be expressed and induce immediate cell death.
The three major steps in DNA cloning
a) Removing DNA from a source
b) Placing that piece into a cloning vector
c) introducing the recombinant vector into a different cell for expression and/or amplification
The two best ways to remove a piece of DNA from a genome
Restriction enzyme digestion
PCR amplification
physically cut it out of the genome and then purify it
Restriction enzyme digestion
amplify one region and then purify it via gel electrophoresis
PCR amplification
are generally unstable and easily degraded
linear fragments of DNA
where do we have to insert linear fragments of DNA
insert them into a more stable shuttling vector prior to manipulating them and/or moving them in and out of cells.
are small, circular pieces of DNA that have the ability to exist and replicate separately from the host genome
cloning vectors
The most commonly used cloning vectors are
plasmids
why are plasmids common
because of their small size, availability, and ease of use.
Plasmids, which were originally isolated from bacteria, can hold DNA fragments that are
10,000 bp or less
In order to be effectively used as a cloning vector, plasmids have to
contain restriction enzyme cut sites that are only found once within the plasmid.
the two pieces can then be ligated together with the help of
DNA ligase
restriction enzyme cut site used
HindIII
plasmid cloning vector we are using in lab
pUC19
