Describe the five general procedures in DNA cloning.
- Obtain DNA segment to be cloned
- Select the cloning vector
- Join the two DNA fragments w/ DNA ligase to form recombinant DNA
- Move recombinant DNA to host organism
- Select/Identify the cells w/ your target DNA
Are restriction enzymes made by eukaryotes or prokaryotes?
Prokaryotes
What is the function of restriction enzymes in vivo?
To recognize + cleave foreign DNA (ex: viral DNA)
Discuss the restriction-modification system.
- Restriction endonucleases cleave DNA at specific sequences
- Sequences within the organism’s own DNA that match those recognized by restriction enzymes are protected by methylation.
What is a restriction endonuclease that generates sticky ends and one that generates blunt ends?
Sticky End Enzymes = BamHI, ClaI, EcoRI, HindIII, NotI, PstI, Tth111I
Blunt End Enzymes = EcoRV, HaeIII, PvuII
What are blunt and sticky ends?
Sticky ends: Two overhanging DNA ends w/ unpaired nucleotides as a result of staggered cleavage by a restriction endonuclease
Blunt ends: Two DNA ends w/ no unpaired bases – result of straight-across cleavage by a restriction endonuclease
What is a plasmid?
A circular DNA molecule that replicates separately from the organism’s chromosome
What is transformation?
The introduction of exogenous DNA into a cell, causing the cell to acquire a new phenotype
Why do plasmids contain a selectable marker gene?
- The selectable marker gene can be used to select for cells that contain the desired gene.
- Selectable marker either permits the growth of the cell (positive selection) or kills the cell (negative selection) under a defined set of conditions.
Why do plasmids contain an origin of replication (ori)?
- The origin of replication is a sequence to which cellular enzymes can bind to initiate replication.
- Necessary for the propagation of the plasmid.
Why do plasmids contain unique recognition sequences for restriction endonucleases?
- The recognition sites provide areas where the DNA can be cleaved for the insertion of foreign DNA.
How are plasmids used to clone DNA?
- The plasmid is cleaved by restriction enzymes
- DNA fragments to be cloned are ligated (joined) to the cleaved plasmid by DNA ligase
- Cells incorporate the plasmid + are transformed
- Cells are grown under certain conditions to determine which ones took up the plasmid
- The plasmid is replicated within the cell.
What is an expression vector?
A cloning vector w/ the transcriptional + translational signals needed for the regulated expression of a cloned gene.
Why do you need an expression vector to express a eukaryotic gene in a prokaryote?
Eukaryotic genes lack the DNA sequence elements necessary for expression in prokaryotic cells (promoter for RNA polymerase binding, ribosome-binding sites) +
What is the importance of the origin of replication (ori)?
DNA sequence to where replication of the plasmid is initiated.
What is the importance of the selectable genetic marker?
Gene used to identify which cells incorporated the plasmid (ex: antibiotic resistance gene).
What is the importance of the polylinker?
Contains restriction sites where, once cleaved, the desired gene can be inserted into the plasmid.
What is the importance of the promoter?
DNA sequence to direct RNA polymerase binding for mRNA synthesis.
What is the importance of the operator (O)?
DNA sequence to which a repressor protein can bind, regulating RNA synthesis.
What is the importance of the ribosome-binding site?
Provides sequence signals for efficient translation of mRNA synthesized from the gene.
Why is it necessary to have a promoter in the expression vector when using a cDNA inserts?
So that genes inserted at restriction sites have regulated expression. Most eukaryotic genes lack the instructions for RNA polymerase that they need to work in prokarya.
Why is it desirable to have a regulatable promoter in an expression vector?
- Rate of expression controlled by replacing the gene’s own promoter + regulatory sequences w/ more efficient + convenient versions leading to high expression of the cloned gene
o High efficiency leads to overproduction of the cloned gene’s protein product– at high concentrations of cloned gene, foreign proteins can kill the cell
o If promoter is able to be regulated, expression of the cloned gene can be regulated.
Define site-directed mutagenesis.
- A set of methods used to create specific alterations in the sequence of a gene
- Alter DNA sequence of cloned gene to change amino acid sequence of resulting protein (way to study protein structure + function)
Distinguish the synthetic insert method from oligonucleotide directed mutagenesis.
- Site-directed mutagenesis: cut out DNA + insert synthetic DNA w/ altered nucleotide sequence
- Oligonucleotide directed mutagenesis:
o Two complementary oligonucleotide sequences act as primers for synthesis of mutant plasmid
