Biology A2 Chapter 21 - Recombinant DNA technology Flashcards
Define recombinant DNA
When the DNA of two different organisms has been combined
What is a transgenic/genetically modified organism?
One which has recombinant DNA
What are the stages of making a protein using the DNA technology of gene transfer and cloning? Give a brief explanation of each
- Isolation - isolate the DNA fragments that have the gene for the desired protein
- Insertion - insert the DNA fragment into a vector
- Transformation - transfer the DNA into suitable host cells
- Identification - identify the host cells that have successfully taken up the gene by the use of gene markers
- Growth/cloning - clone the population of host cells
What are the three methods of producing DNA fragments?
- Conversion of mRNA to cDNA using reverse transcriptase
- Using restriction endonucleases to cut fragments containing the desired gene from DNA
- Create the gene in a gene machine
Describe the process of using reverse transcriptase to isolate a gene using insulin as an example
- B-cells from the islets of Langerhans in the human pancreas are required as they are specialised to produce insulin and so make a lot of mRNA which codes for insulin
- The mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
- cDNA is isolated by the hydrolysis of the mRNA with an enzyme
- Double stranded DNA is formed on the cDNA template using DNA polymerase
- A copy of the human insulin gene is produced
Describe the process of using reverse transcriptase to isolate a gene
- A gene which readily produces the protein is selected
- These have large quantities of the relevant mRNA and so it is easily extracted
- Reverse transcriptase is used to make DNA from RNA. This is known as cDNA
- DNA polymerase forms the double strand of DNA required
What is a recognition sequence?
The particular sequence of bases where a restriction endonuclease cuts
What are the two ways in which restriction endonucleases cut DNA?
- Blunt ends - cut to leave two straight edges, cut between two opposite base pairs
- Sticky ends - an even cut where each strand of the DNA has exposed bases
Describe the process of using the gene machine to isolate a gene
- The desired sequence of bases is determined from the protein. The mRNA codons are worked out from the amino acid sequence and complementary DNA triplets are also worked out
- The desired base sequence is fed into a computer
- This is checked for biosafety and biosecurity reasons
- The computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene
- This gene is then replicated using using PCR which also contracts the complementary stand of nucleotides to make the double stranded gene. This is then multiplies to give numerous copies
- Using sticky ends the gene can be inserted into a plasmid. This acts as a vector for the gene, allowing it to be stored, cloned, or transferred into other organisms in the future
- The genes are checked using standard sequencing techniques and those with errors are rejected
What is meant by in vivo?
Transferring the fragments into a host cell using a vector (in life)
What is meant by in vitro?
Using the polymerase chain reaction (PCR), so it is done ‘in glass’
What is the purpose of the enzyme DNA ligase?
Binds the phosphate sugar framework of the two sections of DNA to unite them as one
Why are sticky ends important?
Provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism
Define the term promoter
The region of DNA which acts as a binding site for RNA polymerase and transcriptional factors, starting transcription
Define the term terminator
The region of DNA which releases RNA polymerase, ending transcription
Define the term vector
The carrying unit to which the DNA fragment is added
What is a vector used for?
To transport the DNA into a host cell
Why will not all the bacterial cells possess the DNA fragment with the desired gene for the desired protein?
- Only few bacterial cells (1%) take up the plasmids when the two are mixed together
- Some plasmids will have closed up again without incorporating the DNA fragment
- Sometimes the DNA fragment ends join together to form its own plasmid
How do we find out which bacterial cells have taken up the plasmid containing the DNA fragment? What is the problem with this?
- All the bacterial cells are grown on a medium that contains the antibiotic ampicillin
- Bacterial cells that have taken up the plasmid will have acquired the gene for ampicillin resistance and so they can break down the ampicillin and survive
- The bacterial cells which have not taken up the plasmid will not be resistant and therefore will die
The problem is that some cells which have taken up the plasmids which closed before incorporating the new gene, and these would also have survived
How do we use marker genes to identify whether a gene has been taken up by bacterial cells?
We use a second, separate gene on the plasmid
Why may certain genes on plasmids be easily identifiable using marker genes?
- May be resistant to an antibiotic
- May make a fluorescent protein which is easily seen
- May produce an enzyme whose action can be identified
How do we identify cells with plasmids that have taken up the desired gene?
Replica plating. The gene was cut to add the DNA fragment and in doing so it lost its resistance to tetracycline as it no longer produces the enzyme to break it down. This means they do not survive against tetracycline and so we can identify living colonies of bacteria containing the desired gene
How do fluorescent markers work?
A gene from a jellyfish is transferred into a plasmid. This produces a green fluorescent protein (GFP). Any plasmid that has taken up the plasmid with the desired gene will not produce GFP and those will did not will produce GFP
How do we use enzyme markers to identify which cell has taken up the desired gene?
If a plasmid with the required gene is present in a bacterial cell, the colonies grown will not produce lactase. Therefore, they will not turn from colourless to blue, and will remain colourless. This is because the required gene to produce lactase was removed from the plasmid when the DNA fragment was inserted
