Chapter 21 - Recombinant DNA Flashcards
What is recombinant DNA technology?
The process by which genes of different organisms can be manipulated, altered and transferred from one another
What is the importance of recombinant DNA technology?
Allows cloning of gene of interest to produce large quantities of the desired protein which can be given to humans to help cure human diseases
What type of cloning uses a vector to transfer a gene into a host cell?
In vivo cloning
How is complementary DNA made using reverse transcriptase?
- Choose a cell that naturally produces the protein so it has a large amount of mRNA which codes for the protein
- Isolate the mrna and reverse transcriptase enzyme and the mrna will act as a template and DNA will join to their complementary base pairs to fomr a single stranded cDNA - complementary DNA
- cDNA is then hydrolysed with an enzyme to remove the mrna
- Double stranded DNA is formed on the template of the cDNA using DNA polymerase
How are restriction endonucleases used to cut out the DNA fragment containing the desired gene of interest?
Restriction endonucleases all have a specific active site complementary to a specific DNA base sequence called the recognition sequence where it cuts at a particular location
What are the two ways that restriction endonucleases can cut DNA?
- Cut at the same location called a blunt end
- Cut staggered to create exposed staggered ends called sticky ends as they cut in a palindromic sequence where top part is the same as the bottom read backwards
Why are sticky ends important?
They have the ability to join to DNA with complementary base pairs
DNA fragments of the sticky ends are complementary to the sticky ends on then plasmid which allows DNA to be inserted into a vector and carried to host cells
How is DNA inserted into a plasmid using restriction endonucleases?
Cut open the vector usually a plasmid using the same one as he one used to create the DNA fragment sticky end
DNA exposed nucleotides on sticky ends are complementary to the sticky ends on the plasmid so enzyme ligase sticks them together by catalysing the condensation reaction to form phosphodiester bonds between adjacent nucleotides
What is the third way you can isolate a DNA fragment?
Creating a gene in a gene machine by manufacturing the gene in a lab
How does a gene machine work?
- The amino acid sequence of the protein is determined
- The mRNA codons are found
- From this the DNA triplets are worked out
What is the most common way to isolate a DNA fragment?
Restriction endonucleases
How is the DNA of the vector introduced into the host cell?
Cell membrane is made more permeable to allow the vector to pass through this is done by adding calcium ions or using heat shock
What are the three outcomes when bacteria is mixed with plasmids (vectors) ?
- 99% not transformed as they don’t take up the plasmid
- 0.1% successful transformation the plasmid is taken up but vector doesn’t contain gene of interest
- Very few are successfully transformed and transgenic which means they have taken up the plasmid ad contain the gene of interest
What are the 3 main reasons why bacterial cells are not transformed?
- Recombinant plasmid didn’t get inside the host cell
- Plasmid re joins with itself before the DNA fragment entered so no sticky ends for the DNA to bind with
- DNA fragment sticks to itself rather than inserting into the plasmid
Why do we use bacterial cells?
They rapidly divide by binary fission
