gene cloning
the procedure of isolating and making many copies of a gene
One typically clones chromosomal DNA into a
DNA vector
what serves as the source of the DNA segment of interest
chromosomal dna
Describe vector DNA
- serves as the carrier for the DNA segment that is to be cloned
- can replicate independently of the host chromosomal DNA
vectors used in gene cloning are usually derived from:
naturally occurring plasmids, but sometimes viruses
describe plasmid vectors
- High copy number bacterial plasmids give higher DNA yields when DNA is purified (limit: ~20-kb inserted)
- Low copy number bacterial plasmids can handle having larger DNA fragments inserted into them when a bacterial chromosomal origin of replication is used: 100-kb to 200-kb can be inserted.
viruses
have sequences removed or mutated so that it is no longer a pathogen but can still function as a vector
commercially available plasmids have
selectable markers
genes conferring antibiotic resistance to the host cell most commonly include
Ampicillin resistance (AmpR)
Chloramphenicol resistance (CanR)
Kanamycin resistance (KanR)
Typical bacterial plasmid vector design
usually grown in E. coli
host cell
the cell that harbors the vector
- DNA the vector carries gets replicated
bacterial host cell
- The host cell is usually a non-pathogenic strain of the bacterium E. coli
- The bacterial host cell lacks the antibiotic resistance that the plasmid has so the bacterium only survives an antibiotic if a plasmid is present
Yeast Host Cell
Yeast can be used as a eukaryotic host cell but yeast has a tendency to rearrange (alter) the cloned DNA
Also: antibiotics cannot be used to retain plasmids in yeast. Instead, people have an essential gene exclusively on the plasmid
to prepare chromosomal DNA; the scientist has to:
- obtain cellular tissue and break open the cells
- extract and purify DNA using a variety of biochemical techinques
- obtain the DNA fragment of interest
restriction enzymes
- used by bacteria as a defense against viruses
- many bacteria methylate their own DNA at specific sites
- the bacteria also express restriction enzymes that would cleave the DNA if it were unmethylated
- viruses that infect the bacteria have unmethylated DNA and can get cleaved (unless virus found a way around this problem)
Many cloning experiments involve restriction enzymes (a.k.a. restriction endonucleases)
- restriction enzymes bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand
- some restriction enzymes produce “blunt ends” while others produce “sticky ends”
Ex. BamHI or Hpal
using restriction enzymes that produce sticky ends to cleave and ligate
- DNA from two different sources incubate both with EcoRI which cuts the DNA
- incubating together H bonds the two sticky ends
- add DNA ligase which covalently links the DNA backbones
typical bacterial plasmid vector design
LacZ encodes a protein (beta-galactoside) that can cleave a chemical called “X-gal”
- intact X-gal is colorless
- cleaved X-gal is blue
Four ways to clone a gene
- Shotgun clone genomic DNA into a vector and identify gene of interest (best way, but most work)
- Make a cDNA library and shotgun clone this into a vector and then identify gene of interest
- PCR amplify the gene and clone it into a vector
- RT-PCR a gene and clone it into a vector
Describe shotgun clonning
Ex. human b-globin gene
1. cut the human and plasmid DNAs with the same restriction enzyme
2. mix DNAs together
3. sticky ends base pair
4. DNA ligase covalently links
5. mix DNA with E. coli cells that have been treated with chemicals to make them permeable
6. plate cells on media containing X-gal, IPTG (allolactose mimic), and ampicillin and incubate overnight
7. each colony is derived from a single cell with a different plasmid
8. filter is gently laid onto the master plate and lifted so the colonies stick
9. filter is treated with detergent to permeabilize the bacteria & DNA is fixed to the filter with UV light
10. NaOH is added to denature the DNA
11. a probe is added that is complementary to the b-globin gene
12. filter is washed to remove unbound probe and placed next to x-ray film
13. identify colonies from orientation of the master plate
blue colony
recircularized plasmid without insert
white colony
hybrid vector with an insert
disadvantages of genomic DNA cloning
- gene may be too large for a plasmid (BACs may be used instead)
- more difficult to clone large DNA fragments
advantages of genomic DNA cloning
- also have the promotor
- gene in genomic DNA often is more accurately expressed if make transgene from it (alternatives)
