Recombinant DNA Technology Flashcards
A RECOMBINANT DNA MOLECULE
A molecule of DNA that has originated from 2 or more DNA fragments that are
not found together in nature
Steps in cloning a DNA fragment of interest
CLONING
Production of identical copies of a particular DNA molecule
Isolation of a particular piece of DNA from the rest of a cell’s DNA
Cloning DNA using restriction enzymes
- Cloning DNA can be used for example to make many copies of a DNA fragment, to produce proteins in bacteria or to study gene mutations
- Plasmids and DNA that should be inserted into the plasmid can be cut using restriction enzymes
-This produces complimentary ends - Restriction enzymes are sequence specific
- Mix together:
-Cut plasmid - Cut DNA fragment
-DNA ligase
-ATP - Resulting DNA is a circular plasmid that now includes the introduced DNA
Restriction Enzymes - What are they?
> Restriction enzyme = Endonucleases = endo DNases
* Digest double-stranded DNA at internal phosphodiester bonds
Cut DNA at specific sites - shown is a site for the enzyme EcoRI
* defined by nucleotide sequences = recognition sequence = restriction sites = restriction sequences
: Panceoides (some have more)
> Leave 3’-hydroxyl and 5’-phosphate groups on both cut strands
Palindromic DNA sequences as restriction enzyme recognition sites photo
Restriction Enzymes - What are they? photo
Restriction Enzymes - Naming
Restriction Enzymes can produce different DNA ends photo
Restriction enzymes - Not a human invention photo
Plasmids - what are they?
Small, extrachromosomal, double-stranded, circular DNA molecules
: Naturally round in bacterial cells
* Carry genes that are beneficial for the survival of the bacterium under certain conditions - e.g. antibiotic resistance.
* Distinct from the bacterial chromosome - replicates independently from chromosomal DNA
* Also called “Vectors” → move DNA from one place to another
Plasmids have many features
- Promoter: allows for expression of a gene cloned downstream of the promoter
- Restriction enzyme sites: usually restriction sites that occur only ones in the plasmid. Allow for cloning of a DNA of interest. Otten there are many
unique restriction enzyme sites = multiple cloning
site or polylinker - Origin of replication: allows for replication of the plasmid to ensure that it is past on to daughter cells
O2
plasmid are tound in a bacteral cell - Selection marker: the selectable marker is used to select for bacteria that have the plasmid. If the marker is an antibiotic resistance gene, then bacteria without the marker will die in the presence of the antibiotic.
Plasmids have many features photo
DNA ligase photo
Insertion of a DNA fragment into a plasmid photo
Summarising the cloning of DNA using restriction enzymes
in a short video
- Cloning DNA can be used for example to produce proteins in bacteria
- Plasmids and DNA that should be inserted into the plasmid can be cut using restriction
enzymes - This produces complimentary ends
- Restriction enzymes are sequence specific
Video from: hitos-/ww.woutube.com/watch?valk Pyhitl.M0 - Mix together:
-Cut plasmid - Cut DNA fragment
- DNA ligase
-ATP - Resulting DNA is a circular plasmid that now includes the introduced DNA
A typical plasmid (vector) map photo
Transformation of bacterial cells photo
Review of the steps in cloning a DNA
fragment of interest photo
Agarose Gel Electrophoresis photo
Agarose Gel Electrophoresis photo
Agarose Gels
.
Agarose is a red algal carbohydrate
ooll in outer to dissolve
Poor into a tray containing a comb
Forms a solid gel with loading pockets when it
Cools down
* Pull out combs, transfer tray with gel into an electrophoresis tank and overlay with a buffer
Overexpressed proteins are purified by affinity chromatography photo
Large scale production of proteins
Host cells
* bacteria
* yeast cells
* mammalian cells
insect cells
Expression vector encodes highly active promoter region
* peptide tag for isolation of expressed protein, e.g. the nucleotide sequence coding for a cluster of histidine residues (His-tagged)
* A cluster of histidine residues can bind to metal ions (next slide)
Large scale production of proteins photo
