21. rDNA TECHNOLOGY Flashcards
Define what recombinant DNA is
Recombinant DNA contains DNA from two types of organisms
Which feature of DNA means an organism from one species will be able to transcribe and translate DNA from a different species?
DNA is universal
Describe the three ways the desired gene can be isolated
Using restriction endonucleases to cut existing DNA, using reverse transcriptase to convert mRNA into cDNA, and production of the gene using a gene machine
What are restriction endonucleases?
Enzymes that cut DNA at specific restriction sequences / recognition sites
Why would two different restriction endonucleases be required to isolate the desired gene from an existing genome?
Because the restriction site at the beginning and the end of the gene would be different
Restriction endonucleases leave sticky ends. What are sticky ends?
Sticky ends are short sequences of bases on the end of DNA that are not bonded to their complementary base pair
Why can you not isolate a gene to be inserted into a bacteria using restriction endonucleases?
Because the gene would include the introns, and bacteria cannot splice out introns.
What is reverse transcriptase?
An enzyme that produces DNA from mRNA
If using reverse transcriptase to produce DNA, which cells would you choose to use?
Cells already translating the protein, as they should contain lots of the mRNA for that gene.
Why is it better to isolate a gene to be inserted into bacteria using reverse transcriptase rather than restriction endonucleases?
Because mRNA does not contain introns, therefore the gene produced from it will not contain introns either
What is a gene machine?
A computer that can reverse engineer a gene from a known protein
Describe a benefit of isolating a gene using a gene machine
It’s faster
Which process is used to amplify (make more copies of) the isolated gene?
PCR (Polymerase Chain Reaction)
Describe the function of PCR
PCR is used to increase the amount of DNA when there is only a small amount in a sample
Apart from amplifying gene fragments for rDNA, describe another use of PCR
To amplify DNA from a crime scene to ensure there is enough to carry out genetic fingerprinting
Which type of cloning is PCR an example of?
In vitro
List the ingredients required for PCR
Sample of DNA to be amplified, primers, DNA Polymerase, DNA nucleotides
Name the machine that PCR is carried out in
A thermocycler
What are primers?
Short fragments of single-stranded DNA which have a specific base sequence which is complementary to a section of the gene to be amplified / copied
Describe the function of primers
They allow DNA polymerase to attach to and copy the correct gene
Why are two different primers required for PCR?
Because the sequences at the beginning and end of the gene are different
Describe the function of the DNA nucleotides in PCR
To form the new DNA strands
Why is the temperature of the thermocycler increased to 95 degrees?
To break the hydrogen bonds between the complementary base pairs. This separates the strands to produce single strands.
After the primers and nucleotides are added, the thermocycler is cooled down to 70 degrees. Explain why.
To allow the reformation of the hydrogen bonds, so the primers and free nucleotides can bond to their complementary base pairs
Describe the function of DNA polymerase
To catalyse the formation of phosphodiester bonds between adjacent nucleotides to produce a complementary strand of DNA
Why is a heat stable version of the enzymes used?
So they don’t denature at high temperatures
Why might the number of DNA molecules produced from PCR plateau, even though the thermocycler is still completing cycles?
The free nucleotides are used up, so no new DNA strands can be made
Which type of cloning is rDNA an example of?
In vivo
What is a vector?
Vectors are carriers of DNA into a host cell
What are most commonly used as vectors to insert the desired gene into the host cell?
Plasmids
When inserting the desired gene into a plasmid, why should the plasmid and the gene be cut with the same restriction endonucleases?
To ensure the gene and the vector (plasmid) have the same sticky ends that are complementary
Describe the function of DNA ligase in the insertion of the desired gene into the plasmid
DNA ligase joins the complementary sticky ends of the vector and the gene by forming phosphodiester bonds
Apart from the desired gene, what two other pieces of DNA might be added to the plasmid?
Promoters and markers
Describe the function of promoters
Promoters ensure the protein is only translated in certain target cells
When are promoters used?
When the rDNA is being inserted into a multicellular organism that contains different types of specialised cells.
Explain why its important to ensure the desired gene is only expressed in target cells?
Translation of the gene in non-target cells could be dangerous
Describe the function of markers
Markers are used to identify which host cells have been transformed
Explain why its necessary to use markers to identify transformed cells
Not all plasmids will take up the desired gene, and not all host cells will take up a plasmid
Describe the three possible outcomes of the mixing of plasmids and the desired gene
The desired gene is taken up into the plasmid.
The plasmid closes up without the desired gene being incorporated.
The gene fragments join together without being incorporated into a plasmid.
Name the three most commonly used markers
A gene that codes for a GFP (green fluorescent protein)
A gene that codes for antibiotic resistance
A gene that codes for an enzyme
Why is using the GFP gene as a marker efficient?
Because the transformed bacteria can be identified quickly using the UV light
How would transformed bacteria be identified if the antibiotic gene marker was used?
The bacteria would be plated on agar containing the bacteria. The cells that don’t die are the transformed cells
If the desired gene is being inserted into a multicellular organism, how would you ensure all the cells of the organism contain the gene?
Insert it into embryonic cells. Then when these divide by mitosis to produce genetically identical cells, they will contain the desired gene (as its also replicated)
Probes can be used to identify if certain alleles exist in a persons’ genomes. Give three reasons why someone might want to see if their genome contains a certain allele.
To find out if they (or their partner) were a carrier of a genetic disease.
To find out if they have an oncogene.
To find out if they have an allele that would increase their chances of developing a genetic condition later in life.
What is a probe?
A piece of single stranded DNA with bases complementary to a known allele
What are the two ways probes can be labelled?
Under UV light. The sample will glow if the probe is present.
How are radioactive probes labelled?
By using the sample to expose an X-Ray film. Dark bands will appear if the probe is present.
What must be the bases on the probe be complementary to?
The allele you are trying to detect
How are many copies of the probe produced?
PCR
Why is the patient’s DNA heated?
To break the hydrogen bonds between complementary base pairs to produce single strands
After the probes are added, why is the temperature reduced?
To allow the formation of hydrogen bonds between the probe and the allele on one strand of the patient’s DNA (if it exists)
Why is the mixture washed?
To remove any unbonded probes
A probe is used that is complementary to a dominant allele. How many probes should bind to a heterozygous person’s DNA compared to a homozygous dominant person’s DNA?
Half the amount of probe should bind to a heterozygous person’s DNA compared to a homozygous dominant person’s DNA
What is gel electrolphoresis?
Gel electrophoresis is a method used to separate DNA fragments according to their size
Describe three uses of gel electrophoresis
Identify the presence of a known gene in a genome.
Identify the strain of pathogen infecting a patient.
Genetic fingerprinting.
Describe three uses of genetic fingerprinting
Paternity testing.
Identifying suspects in a crime.
To reduce inbreeding when breeding captive animals.
What is used to cut the DNA sample before separation?
Restriction endonucleases
What is passed over the DNA sample after it’s put into the wells in the gel?
An electric current
Describe and explain how the DNA fragments will separate
The DNA fragments will separate according to size. This is because the negative DNA moves towards the positive electrode. Smaller pieces of DNA move further.
What is added to the gel to make the DNA single stranded?
An alkali
What is the DNA sample transferred onto?
A sheet of nylon
Why is the DNA made to be single stranded?
So when the probes are added, they will bind to the DNA by complementary base pairing
Genetic fingerprinting is carried out by analysing the number and size of what type of DNA in genomes?
VNTRs
What are VNTRs?
Variable Number Tandem Repeats. They are repeats of short sections of bases that are found in the introns of DNA.
When analysing how closely related two people are, why should you cut their DNA using the same restriction endonucleases?
So their DNA is cut at the same points (at the beginning of the same VNTRs)
What will the probes used to visualise their DNA be complementary to?
The VNTRs
How is relatedness ascertained using genetic fingerprinting?
The more similar the pattern (the more bands in common), the more closely related the two individuals are.
How can paternity be decided using genetic fingerprinting?
50% of the child’s genetic fingerprint bands should be the same as the mother’s and the other 50% should be the same as the father’s.
How can a suspect be identified from the DNA left at a crime scene?
The genetic fingerprint of the suspect should exactly match that found at the crime scene.
How can genetic fingerprinting be used to reduce inbreeding when breeding captive animals in zoos?
The individuals should be as distantly related as possible, so their genetic fingerprints should have the fewest bands in common as possible.
