Recombinant DNA technology Flashcards
What is recombinant DNA technology?
- transferring a fragment of DNA from one organism to another
Why is recombinant DNA technology possible?
- The genetic code is universal and so are transcription and translation mechanisms
- Therefore, the transferred DNA can be used to produce a protein in the cells of the recipient organism as it can be translated
What is the process of making DNA?
- Isolation = of the DNA fragments that have the gene for the desired protein
- Insertion = of the DNA fragment into a vector
- transformation = the transfer of DNA into suitable host cells
- Identification = that the host cell has been taken up by using gene markers
- growth/cloning = of the host cells
What are the methods in which DNA can be identified and isolated?
- conversion of MRNA to CDNA using reverse transcriptase
- using restriction endonucleases
- creating the gene in a gene machine
Describe method 1, using reverse transcriptase?
- A cell that readily produces the protein is selected
- these cells have a large quantity of MRNA which is then extracted
- reverse transcriptase is then used to make DNA from RNA
- This DNA is known as complementary DNA as it is made of nucleotides which are complementary to MRNA
- CDNA is then isolated by the hydrolysis of MRNA using an enzyme
- double stranded DNA is formed by the template of CDNA using DNA polymerase
Describe method 2, using restriction endonuclease?
- use the same restriction endonuclease to cut the DNA at a specific recognition site and the plasmid
- this will leave complentary sticky ends
What are restriction endonucleases?
enzymes that recognise specific palindromic sequences and cut DNA at these places
- some cut between two opposite base pairings and leave two straight ends whereas others cut unevenly and leave a staggered cut of sticky ends
What are palindromic sequences?
sequences that consist of antiparallel base sequences that can be read in opposite directions
What is a gene machine?
- technology that has been developed so that fragments of DNA can be synthesised without needing pre - existing DNA templates
What is the process of using a gene machine?
- the desired nucleotide bases are fed into the computer
- the computer designs small overlapping single strands of nucleotides called oglionucleotides which can be assembled into the desired gene
- each of the oglionucleotides is assembled by adding one nucleotide at a time in the required sequence
- the oglionucleotides are then joined, forming a gene that does not contain introns
- the gene is the replicated in the polymerase chain reaction
- the polymerase chain reaction also constructs the complementary strand of nucleotides to make the double stranded gene
- using sticky ends, it is then inserted into a bacterial plasmid acting as a vector
What are the two types of cloning?
in vivo = gene copies are made inside of a living organism. as the organism grows and divides it replicates the DNA and produces clones
in vitro = gene copies are made outside of a living organism using the polymerase chain reaction
What is the importance of sticky ends?
- if restriction endonucleases cut the recognition site in a staggered fashion, it leaves single strands with a few nucleotide bases at the end of each DNA single strand
- if the same restriction endonuclease is used to cut DNA, the fragments have complementary sticky ends and can therefore be joined by DNA ligase
How do you prepare the DNA fragment for insertion?
- for transcription of any gene to occur, RNA polymerase must bind to the gene. The binding site of RNA polymerase is called a promoter reigon
- to ensure the gene is transcribed, a promoter reigon must be added and also a terminator reigon to ensure that RNA polymerase is released and ends transcription
What are the steps of making recombinant DNA?
- the vector DNA is isolated
- the vector DNA is cut open using the same restriction endonuclease as the target DNA fragment. This means that the sticky ends of the plasmid and DNA will be complementary
- The vector and DNA fragment are therefore joined using DNA ligase which joins the sticky ends together
How do you transform cells?
- the vector with the recombinant DNA can then be used to transfer the gene into host cells
- host cells that take up the vector are said to be transformed
- This can be done in a solution of calcium ions which change the temperature and increase the permeability of the bacterial cell
Why are all cells not transformed?
- only a few bacterial cells will take up the plasmid
- some plasmids will close up without taking up the DNA fragment
- The DNA fragment may rejoin and form its own plasmid
How can you identify transformed cells?
- marker genes can be inserted into vectors at the same time the gene is cloned. This means any cloned cell will contain the marker gene
- Host cells are grown on agar plates and divide, replicating DNA and produce cloned cells. Transformed cells will produced colonies where all the cells contain the cloned and marker gene
- The marker gene may code for antibiotic resistance, therefore cloned cells are grown on an agar plate that contains the antibiotic. Those that survive are transformed
- The marker gene may code for flouresence and when UV light is shone, will glow in the dark
