Lecture 19: Recombinant DNA technology Flashcards
What are recombinant DNA technologies?
Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce (express) a useful protein.
What is the crucial element in recombinant DNA technologies?
Plasmids
What are the key components of Plasmids?
OAPMR (Orange Apples Pears Melon Retard)
Origin of replication (ORI)
* allows initiation of replication using host DNA polymerase
Antibiotic resistance gene
* Provide survival advantage to cells containing plasmid.
Promoter
* Drives expression of your favourite gene (e.g., insulin or
GFP) in cells with appropriate transcription factor machinery
Selectable Marker
* to select for cells that have successfully taken up the plasmid. For example, GFP gene.
Restriction Sites
* allows ligation of gene of interest into the cloning vector.
What is a plasmid?
- Circular pieces of double stranded DNA.
- Replicate independently of the host’s chromosomal DNA.
- Common in bacteria (but also found in eukaryotes).
- Provide a benefit to hosts e.g., antibiotic resistance.
What are DNA ligases?
DNA ligases are enzymes that join together strands of DNA by forming a covalent bond between the sugar-phosphate backbones of the DNA strands. They are essential in DNA replication, repair, and recombination, helping to seal nicks or breaks in the DNA molecule.
How are restriction enzymes and DNA ligases useful in recombinant DNA technologies?
Restriction enzymes act as molecular scissors, cutting DNA at specific sequences to create fragments. DNA ligases then act as molecular glue, joining these fragments together. This allows scientists to insert genes or DNA sequences from one organism into another
Briefly outline the process of transformation: TAEA
TAEA (Take All Eggs Away)
- Transformation = transfer of vectors into bacteria
- Transformed bacteria selected by antibiotic resistance contained on the recombinant vector.
- Expression of vector gene in bacteria (if bacterial promoter).
- Amplification of bacteria and purification of DNA for downstream uses e.g. PCR, cloning, transfection into other cells or organisms.
How is recombinant DNA technology made possible via the genetic code?
- All organisms ”read” the same codons as the same amino acids.
Therefore we can transform a human gene into bacteria, and it will still make the same protein.
What issue may be encountered when cloning eukaryotic genes in prokaryotes?
Prokaryotic genes don’t have introns (like eukaryotes) and therefore cannot cut them out.
This would lead to a nonsense polypeptide that did not fold into the correct tertiary structure
How is cloning eukaryotic genes in prokaryotes made possible?
By Using the coding sequence only! (cDNA)
cDNA is generated via reverse transcriptase
No introns allows successful translation to a functional protein in prokaryotic cells.
Without the intronic sequence, the overall size of the insert is reduced. – Many vectors have insert size restrictions
What is the universal genetic code and why is it necessary for recombinant DNA technologies?
Every organism reads 3 letter codons as the same amino acids, this is important as we can therefore for example use bacteria to make human proteins
Name two components required to create a plasmid to express a human gene in bacteria:
(Any 2 of)…
An origin of replication - allows initiation of replication using host DNA polymerase
A promoter region - Drives expression of your favourite gene (e.g., insulin or GFP) in cells with appropriate transcription factor machinery
A selectable marker - to select for cells that have successfully taken up the plasmid. For example, GFP gene.
Antibiotic resistance - Provide survival advantage to cells containing plasmid.
Restriction cut sites - allows ligation of gene of interest into the cloning vector.
