21. Recombinant DNA Technology Flashcards
What are many human diseases the result of?
Individuals being unable to produce for themselves various metabolic chemicals. Many chemicals are proteins, and therefore the product of a gene.
What are the disadvantages from producing large quantities of ‘pure proteins’ from other sources?
- Rejection from immune system
* Considerable cost
What are the advantages from producing large quantities of ‘pure proteins’ from other sources?
Being able to cure diseases.
Improved quality of life.
Prolonged lifespan
What is ‘recombinant DNA’?
The DNA of 2 organisms that has been combined
What is the resulting organism called which has recombinant DNA?
Transgenic/genetically modified organism.
Describe the process of making a protein using gene transfer technology?
- Isolation of the DNA fragments that have the gene for the desired protein.
- Insertion of the new DNA fragment into a vector.
- Transformation of DNA into suitable host cells.
- Identification of the host cells that have successfully taken up the gene, by gene markers.
- Growth/cloning of the population of host cells.
Give examples of methods used to produce DNA fragments
- Conversion of mRNA to cDNA using reverse transcriptase.
- Using restriction endonucleases to cut fragments containing the desired gene from DNA.
- Creating the gene in a gene machine which is usually based on a known protein structure.
Describe the process of making DNA from RNA using reverse transcriptase
- A cell that readily produces the protein is selected (e.g. the β-cells from islets of Langerhans from the pancreas produce insulin)
- These cells have large quantities of the relevant mRNA, which is therefore more easily extracted.
- Reverse transcriptase is then used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it’s made up of the nucleotides that are complementary to the mRNA.
- To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene.
What is a restriction endonuclease?
Enzymes that bacteria use to defend themselves by cutting up viral DNA.
How do restriction endonucleases produce DNA fragments?
Each enzyme cuts a DNA double strand at a specific sequence of bases called a recognition sequence. Sometimes, this cut occurs between 2 opposite base pairs. This leaves 2 straight edges known as blunt ends. Others cut DNA in a staggered fashion. This leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases. If you read 2 sequences of unpaired bases that are opposites of one another they are a palindrome. This is typical of the way restriction endonucleases cut DNA to leave sticky ends.
Describe how the ‘gene machine’ produces DNA fragments
•. The amino acid sequence of this protein is determined. From this, the mRNA codons are looked up and the complementary DNA triplets are worked out.
• The desired sequence of nucleotide bases for the gene is fed into a computer.
• The sequence is checked for biosafety and biosecurity.
• The computer designs a series of small, overlapping single strands of nucleotides (oligonucleotides) which can be assembled into the desired gene.
• In an automated process, each of the oligonucleotides
is assembled by adding one nucleotide at a time in the required sequence.
• The oligonucleotides are then joined together to make a gene. This gene doesn’t have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction.
• The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double stranded gene. It then multiples this gene many times to give numerous copies.
• Using sticky ends, the gene can then be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organism in the future.
• The genes are checked using standard sequencing techniques and those with errors are rejected.
Give advantages of the gene machine
Any sequence of nucleotides can be produced in short time with great accuracy.
DNA free of introns, so can be transcribed and translated by prokaryotic cells.
Define ‘in vivo’ gene cloning
Cloning by transferring the fragments to a host cell using a vector.
Define ‘recognition sites’
The sequences of DNA cut by restriction endonucleases
Why is it important the same restriction endonuclease is used to cut DNA?
If the same restriction endonuclease is used to cut DNA, then all the fragments produce will have ends that are complementary to one another. This means that the single stranded end of into one fragment can be joined to the single-stranded end of another fragment- their ends are sticky. Once the complementary bases of 2 sticky ends have paired up, an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the 2 sections of DNA together.
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.
How does the DNA fragment need to be prepared before insertion?
For the transcription of any genes to take place, RNA polymerase must attach to the DNA near a gene, to the binding region known as the ‘promoter’. The nucleotide bases of the promotor attach to both RNA polymerase and transcription factors and begin transcription.
Similarly, another region releases RNA polymerase and ends transcription. This region of DNA is known as the ‘terminator’. Both promotor and terminator regions need to be added before DNA insertion.
Describe how DNA is inserted into a plasmid vector
Plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop.
The restriction endonuclease used is the same as the one that cut out the DNA fragment. This ensures that the sticky ends of the opened up plasmid are complementary to the sticky ends of the DNA fragment.
When the DNA fragments are mixed with the opened up plasmids, they may become incorporated into them. The join is made permanent using the enzyme DNA ligase. These plasmids now have ;’recombinant DNA’.
What does ‘transformation’ mean
The incorporated DNA plasmid is reintroduced into host cell. e.g. bacteria.
What makes the membrane of the host cell permeable to the vector?
- Increased temperatures
- Calcium ions
Why might not all of the bacterial cells will possess the DNA fragments with the desired gene for the desired protein?
- Only a few bacterial cells take up the plasmids when the 2 are mixed together
- Some plasmids will have closed up without incorporating the DNA fragment.
- Sometimes the DNA fragment ends join together to form its own plasmid.
Describe how to find out which bacterial cells have taken up the plasmid, using the gene for antibiotic resistance, which is unaffected by the introduction of a new gene.
- All the bacterial cells are grown on a medium containing the antibiotic
- Bacterial cells that have taken up the plasmids will have acquired the gene for antibiotic resistance.
- These bacterial cells are able to break down the antibiotic and therefore survive.
- The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die.
What is the role of marker genes?
Marker genes identify whether a gene has been taken up by plasmids, involves using a 2nd, separate gene on the plasmid.
How do antibiotic resistant genes act as markers to show whether a gene has been incorporated into a plasmid?
Replica plating: This process uses the other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene. The bacteria that has taken up the gene will no longer be resistant to the antibiotic. Problem with testing with antibiotic, such as tetracycline, is that it will destroy the cells containing the required genes, however can use replica technique instead.
How do fluorescent proteins act as markers to show whether a gene has been incorporated into a plasmid?
Transfer of gene from a jellyfish: produces protein (GFP). The gene to be cloned is transplanted into the centre of the GFP gene. Any bacterial cell that has taken up the plasmid with the gene to be cloned will not produce GFP and fluoresce. Bacterial cells that have not taken up the gene will continue to produce GFP and fluoresce.
