Lecture 19 Flashcards
What are recombinant DNA technologies?
Joining bits of DNA together to insert into an organism to produce a useful protein
What are plasmids?
Circular pieces of double-stranded DNA that replicate independently of the host chromosomal DNA and provide benefit to the host e.g. antibiotic resistance
What are the key components of recombinant DNA plasmids (vectors)?
Origin of replication, antibiotic resistance gene, promoter, selectable marker and restriction sites
Why is an origin of replication important?
Allows initiation of replication using host DNA polymerase
Why is antibiotic resistance important?
Provides survival advantages to cells containing plasmids
Why is a promoter important?
Drives expression of genes in cells with appropriate transcription factor machinery
Whys is a selectable marker important?
To select for cells that have successfully taken up the plasmid
Why are restriction sites important?
To allow ligation of gene of interest into the cloning vector
Why do promoters need to be gene spesific?
To allow expression in it, for different cell types
What is a restriction enzyme?
Proteins isolated from bacteria that cut dsDNA at specific sequences - a bacterial defence mechanism
What is DNA ligase?
The enzyme that catalyses the formation of phosphodiester bonds - gluing the two ‘sticky ends’ of the cut plasmid together
What is transformation
The process of introducing recombinant DNA (vectors/plasmids) into bacterial cells
What is the selection process of transformed bacteria?
After transformation, bacteria that have successfully taken up the recombinant plasmid can be selected by growing them on a medium containing an antibiotic. The plasmid contains an antibiotic resistance gene, so only bacteria with the plasmid survive and grow on the antibiotic-containing medium.
What is expression of the vector gene in transformation?
If the plasmid also contains a gene with a promoter that is recognised by the bacterial machinery, the bacteria will express the gene, therefore, producing a protein within the bacterial cell.
What is the amplification and purification of transformation?
The bacteria that have successfully taken up the plasmid will grow and divide, creating many copies of the plasmid and the gene it carries. These bacteria can then be harvested, and the plasmid DNA can be purified. This purified DNA can be used for various applications, such as PCR, cloning, or transfection into other cells or organisms.
What is meant by the term ‘universal genetic code’ ?
All organisms read the same codons as the same amino acids
What is the significance of the universal genetic code?
We can transform a human gene into bacteria and it will make the same protein
What is the limitation of cloning eukaryotic genes for expression in prokaryotes?
Prokaryotes do not have introns and can therefore not preform splicing
What is the solution to the intron problem in prokaryotes?
Use coding sequence DNA only (cDNA) by removing introns before ligating
What is reverse transcription?
The process in cells by which an enzyme makes a copy of DNA from RNA
What does reverse transcription make in relation to pre-prokaryote expression?
cDNA
Why do we use cDNA?
There are no introns, allowing successful translation to a functional protein in prokaryotes and without this intronic sequence, the overall size of the insert is reduces
