Recombinant DNA Flashcards
What is step one of Recombinant DNA?
isolating the gene
What happens when you isolate the gene
Working backwards from the protein – work out the amino acid sequence of the protein wanted, use the genetic code to work out the base sequence and make DNA with that base sequence.
What is another method for isolating the gene?
Using mRNA- use reverse transcriptase enzyme which can synthesize DNA from the relevant mRNA (ie the reverse of the normal process in which mRNA is made from DNA).
What is step two of Recombinant DNA?
Extracting the Gene
What happens when you extract the gene?
. Once the gene is found it must be removed from the DNA of the organism.
. This is done using restriction endonuclease enzymes.
. These enzymes cut the DNA at specific base sequences leaving blunt or staggered (sticky) ends on the DNA sequence they cut.
. The sticky ends readily bond to sticky ends of another DNA sequence cut by the same enzyme.
What happens when you splice the gene into a vector?
Once the gene is removed it is necessary to place it into a cell in which it can be expressed and duplicated so that it passes from one cell division to the next.
A vector is usually required to transfer the foreign gene into bacterial cells.
What is the third step of Recombinant DNA ?
Splicing the gene into a vector
What is the purpose of the plasmid in Recombinant DNA?
. Plasmids (small rings of DNA found in bacteria) are often used as vectors.
The plasmid is cut using the same restriction endonuclease enzyme used to remove the foreign gene.
. The genes are then joined together at their sticky ends using a ligase enzyme.
What is ligation?
When DNA fragments produced using restriction enzymes may be reassembled.
What is annealing ?
When the two matching “sticky ends” come together, they join by base pairing.
What is the fourth step of Recombinant DNA
Introducing the recombinant DNA into a host cell
what happens when you introduce the recombinant DNA into a host cell.
. The recombinant DNA must now be introduced into a host cell, usually bacteria, via the process of transformation
. A transgenic organism is produced.
What happens in the fifth step of Recombinant DNA?
Culturing the host cells
what happens when you culture the host cells?
Bacteria taking up the transferred gene replicate during cell division, producing a clone of genetically engineered bacteria.
The bacterial cells are cultured and produce the gene product in large amounts.
