Lecture 4 Restriction Enzymes and Molecular Cloning Flashcards
Cloning
A collection of molecules or cells, all identical to an original molecule or cell
Make a copy a piece in a plasmid or a whole organism
Ex. Twins are clones
2 very different examples of cloning
- Cell based DNA cloning
2. Cell/organism cloning
Cell based DNA cloning
Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism
Cell/organism cloning
Using nuclear DNA from one organism to create a second organism with the same nuclear DNA
Purpose of cloning?
Determine the sequence of recombinant DNA
To generate a probe for hybridization (Southern blot, Northern blot, etc)
Recombinant expression of the encoded protein and mutagenesis
Regenerative medicine - transplant
Restriction Endonucleases
Restriction Enzymes
Enzymes that cleave DNA molecules at specific nucleotide sequences, called restriction sites
1962 “molecular scissors” discovered in bacteria by Nathans and Smith in 1970
E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA
Restriction Sites
Where enzymes cleave DNA molecules at specific nucleotide sequences
3,000 enzymes have been purified, around 200 have unique properties
General info about restriction endonucleases
Most are 4-8 base pairs
Isolated from bacteria, different types of bacteria so different restriction enzymes
Enzymes that will cut DNA at specific sites defined by the local nucleotide sequence, cleaving a dsDNA into fragments of defined sizes
Most common Restriction Enzyme
EcoRI
Escherichia coli strain R, 1st enzyme
3 Types of Restriction Enzymes
Differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another.
Type I - cuts at random locations
Type II - cut DNA within the recognized sequence without need of ATP
Type III - cuts at approximately 25 base-pairs from the site and also requires ATP (very rare)
Restriction Enzyme Type I
Cuts DNA at random locations as far as 1000 or more base-pairs from the recognition site and requires ATP
-random
Restriction enzymes Type II
Cut DNA within the recognized sequence without the need of ATP
-most used, common because they cut at the recognized restriction site or adjacent
Restriction enzyme Type III
Cuts at approximately 25 base-pairs from the site and also require ATP
-very rare
How Type II Restriction Enzymes work
Cut in close proximity of the recognition site
No ATP requirement
Need Mg2+ as cofactor (in buffer)
Recognition sites in double stranded DNA have a 2 fold axis of symmetry - a palindrome
Cleavage can leave stick end or blunt ends
More than 3500 REs
Most useful for gene analysis and cloning
RE
Restriction Enzymes
Sticky Ends
Also called cohesive since they are easily joined back together by a ligase
A nucleotide fragment can be easily joined with an sticky end vector if digested with the same RE
Allow to do unidirectional cloning
Blunt End Digestion
Vs. Sticky ends
Always compatible with each other
-harder to ligate, less efficient
Disadvantage of potentially inserting the insert DNA in the opposite orientation desired
Plasmids
Naturally occurring extrachromosomal DNA molecules
Circular, double stranded DNA
Can be cleaved by restriction enzymes (sticky or blunt ends)
Can be engineered by linking new DNA fragments to the ends of plasmid
Carries antibiotic resistance cassete and the resistance is often transferred from one bacteria to another
Ori
Origin of replication
Steps of Recombinant DNA
Cut plasmid vector with Aval
Excise DNA insert of interest from source using Ava I
Ligate the insert of interest into the cut plasmid
4 Things important for Cloning Vectors
- Origin of replication - where gene starts
- A selectable marker - antibiotic resistance gene, such as amp^r and ter^r - will grow in medium that has ampicillin and tetracycline
- Multiple cloning site (MCS) - site where insertion of foreign DNA will not disrupt replication or inactivate essential markers
- Might carry a tag (MYC, HA, Flag) or a marker (eGFP)
Origin of replication
Useful for cloning vectors
-where gene starts
Selectable marker
Antibiotic resistance gene on plasmid
Multiple cloning site (MCS)
On plasmid, where insertion of foreign DNA will not disrupt replication or inactivate essential markers
Ex of tags used by plasmids
MYC, HA, Flag
or a marker eGFP
Ligation
The process of joining two pieces of DNA from different sources together through the formation of a covalent bond
DNA ligase is enzyme used to catalyze this reaction
DNA ligation requires ATP
DNA Ligase
The enzyme used to catalyze the reaction of joining two pieces
Ligation of Complementary Ends
Enzymes that cut with staggered cuts result in complementary ends that can be ligated together as long as they are complementary
DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency) but usually cannot be re-cut by either original restriction enzyme
If only one kind of restriction site is used the insert can connect with vector in two orientations
Vector can reconnect with itself without an insert - self ligation
Directional Cloning
Directional ligation requires that two different restriction sites are used at the ends of insert and vector
Identification of Clones and Insert
The clones are evaluated for the presence and the size of an insert and clones with appropriate inserts by using restriction enzymes that cut at sites flanking the insert or within the insert (if known) and gel electrophoresis
- Uncut gel there are bands higher up and make frowny face
- BamHI Digest
Bacteria Transformation
The process of introducing the foreign DNA into the bacterial cell is called transformation
Bacterial act as a host cell so the DNA can be replicated
Not every bacterial cell is able to take up plasmid DNA
Bacterial cells that can take up DNA from the enviro are said to be competent
Two methods for transforming: heat shock and electroporation
Competent
Bacterial cells that can take up DNA from the environment are said to be this
Two methods for transforming
- Heat shock
2. Electroporation
Expression in Mammalian Cells
To produce many copies of a particular DNA sequence, the fragment is first inserted into a plasmid vector. The resulting recombinant plasmid DNA is then introduced into a bacteria, where it can be replicated many millions of times.
Genomic DNA is isolated and purifiied - plasma prep DNA isolation
How are Plasmids Transferred into Mammalian Cells?
- Nucleofection (electroporation) -electrical conductivity changes the permeability of the cell membrane
- Lipofection (calcium chloride, chemical) -chemical that takes advantage of the liposomes. Make holes in cell that allow transfection, not very efficient
- Viral Transduction
- Adenovirus - very effective for high level transient expression. Not stable
- Retroviruses - Very effective for stable expression, only low level expression
Expressing your cloned gene
Even if your plasmid contains insert, it may not be able to generate functional protein from your cloned DNA
- gene may not be intact or mutations that could have been introducted
- the protein encoded by gene may require post-translational modifications
- some enzymes are a complex of peptides expressed from separate genes
Dolly the Sheep
Sheep cloned by nuclear transfer from a cultured cell line
Regenerative Medicine
Bone - osteoblasts Skin Nervous system Liver cells Pancreas - insulin producing cells Heart - cardiomyocytes Vessels - endothelial cells Blood cells Kidney
