DNA technology Flashcards
What is used to produce Human Insulin ?
Genetically modified bacteria
What is the definition of Recombinant DNA ?
DNA that contains (foreign) genes from another organism
What is the definition of Genetically Modified ?
Transgenic organism that contains genes from another organism
What are the different ways the fragment of DNA for genetic engineering can be produced ?
converting mRNA into complimentary DNA (cDNA) using reverse transcriptase
using an enzyme called a restriction endonuclease to cut fragments containing the desired gene from DNA
creating the gene in a gene machine, usually based on protein structure.
Explain why DNA could be inserted into a bacterial cell made from mRNA but not directly from a chromosome
human genes have introns, mRNA does not
bacterial cells cannot remove introns
Explain the use of reverse transcriptase to isolate the gene that codes for insulin
mRNA coding for insulin from Beta cells is isolated and purified. the purified mRNA is mixed with free DNA nucleotides.
mRNA acts as a template on which single stranded complimentary copy of DNA (cDNA) is formed using reverse transcriptase
double stranded DNA is formed on the template strand of cDNA using DNA polymerase
a copy of the human insulin gene is formed.
What is a primer ? what is its function ?
a short length of DNA, 20-30 nucleotides long, which is artificially synthesised to be complimentary to a short section of the single stranded DNA.
the primer acts as a point of attachment for DNA polymerase as it cannot act on single stranded DNA
In what direction does DNA polymerase move along ?
from the 5’ to 3’ end
Explain how a restriction endonuclease can be used to cut the DNA
restriction endonuclease cuts across the DNA molecule
restricition endonucleases break the bonds in the sugar phosphate backbones of both strands of the DNA molecule to produced double stranded fragments
each restriction endonuclease cuts at specific base sequences called recognition sequences (or restriction sites)
most recognition sequences are short
Restriction endonucleases produce an uneven or staggered cut across the DNA molecule. This single-stranded section of the DNA is called a sticky end.
The sticky end will attract and join with complimentary sticky ends
What is the word used to describe recognition sequences ?
palindromic
how are bacterial plasmids cut?
the bacterial plasmid is cut using the same restriction endonuclease
the DNA fragments are then joined by the enzyme ligase
what is in vivo gene cloning using bacteria
the isolated gene is inserted into an appropriate host cell. As the host cell divides it replicates the inserted gene along with its own DNA
Bacteria replicate at a fast rate as they are often used as the host cell- this is in vivo cloning.
What is a vector ?
what are the most commonly used vectors ?
a molecule of DNA which is used to take the donor gene into a microbe
(Most commonly used vectors are
bacterial plasmids)
Explain how the gene is inserted into the plasmid (vector)
the bacterial DNA is cut using the same restriction endonuclease enzyme as that used to cut out the donor gene.
This creates a broken loop of DNA with sticky ends that match the donor gene.
The donor gene and cut plasmid can pair up because their sticky ends have complimentary bases.
They are joined together using the enzyme ligase.
This joins up the sugar phosphate backbone of DNA in a condensation reaction.
What are the various treatments used to make it easier for the plasmids to enter the host bacteria ?
What do these treatments aim to do ?
temperature shock
electrical shock
treatment with ice-cold calcium chloride solution
aim to make the bacterial cell wall more permeable so plasmids can enter the bacteria]
Bacteria that have successfully taken up the plasmid containing the donor gene are said to be what ?
transformed
As the bacteria contain new DNA from a different organism in their plasmid they are said to be what ?
TRANSGENIC
Where do restriction endonucleases cut ?
cut the DNA at specific, palindromic recognition sequences
Describe how antibiotic resistance (marker) genes can be used to identify transformed cells. (describe the method used)
replica plating is used
velvet pad used to transfer bacteria from the surface on one plate to another
one agar plate contains ampicillin
one agar plate contains tetracycline
In bacteria with donor DNA, the tetracycline resistance gene no longer functions, the bacteria are not resistant to tetracycline so are killed
Bacteria with human DNA grow on plate with ampicillin with no tetracycline but are killed on the plate containing tetracycline.
Bacteria with no extra DNA not killed.
The DNA fragments must be modified before they can enter the vector as they will need to be transcribed. Explain what happens during modification
a promoter region is added- at the start of the DNA fragment - allows RNA polymerase to bind to enable transcription to occur.
a terminator region is added- at the end of the gene. It causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA
What is used to identify transformed cells ?
marker genes
why do transformed cells need to be identified ?
not all host cells (usually bacteria) will successfully take up a recombinant plasmid
What are the three different types of marker gene used to identify transformed cells ?
antibiotic resistance genes
genes coding for fluorescent proteins
genes coding for enzymes
Explain how green fluorescent proteins can be used to identify transformed cells
the GFP gene is inserted into the plasmid
DNA fragment is inserted into the middle of the the GFP gene. This disrupts it and prevents GFP production.
grow the colonies on agar and expose to UV light
only non glowing colonies contain the recombinant plasmid
Explain how the enzyme marker lactase can be used to identify transformed cells.
the enzyme lactase can turn a certain substance blue from colourless.
The gene for this enzyme is inserted into the plasmid.
The DNA fragment is inserted in the middle of the gene to disrupt it.
The bacteria are then grown on the agar plate with the colourless substance.
The colonies which cannot turn the colourless substance blue contain the recombinant plasmid
What piece of equipment is used to grow the transformed bacteria on a large scale ?
an industrial fermenter
Explain how the transformed bacteria is cultured.
a fermenter is used to grow multiple copies of the host cell which have been identified as containing the recombinant plasmid.
This large cloned population of the host cell can then produce the protein coded for by the inserted DNA fragment
What does in vivo mean ?
inside of a living organism
Explain why air must be sterilised before it enters the fermenter
to prevent contamination of the culture by harmful bacteria
Explain why nutrients are added to the fermenter
to be used for respiratory substrate
Explain why air is bubbled through an industrial fermenter
for aerobic respiration of the bacteria
What does PCR stand for ?
Polymerisation Chain Reaction
What are the order of stages that take place during In-vivo cloning ?
Isolation of a gene
Insertion into vector (usually plasmid)
Transformation into host cells (plasmid put back into bacteria)
Identification that it has been taken up
Growth
What type of cloning is PCR ?
in-vitro cloning
what does in vitro mean ?
outside of a living organism
list all the equipment used in PCR
thermocycler
DNA fragment to be amplified
DNA polymerase-taq polymerase
primers
DNA nucleotides
Outline the method of PCR
The sample of fragment DNA is mixed with the DNA nucleotides, primers and DNA polymerase in a test tube. The test tube is then placed in a PCR machine (thermocycler)
The mixture is heated to between 94-96 degrees
The hydrogen bonds between the complimentary base pairs break, leaving two single strands of DNA
The mixture is cooled to 50-60 degrees Celsius
At this temperature, the primers will join to template strands by complimentary base pairing at the 5’ end-this is called annealing.
Primers are needed as DNA polymerase involved in the next step has to attach to one of them.
The temperature is increased to 72 degrees Celsius.
Taq polymerase binds to the small section of double stranded DNA (the primer) and synthesises new strands.
Taq polymerase catalyses condensation reactions which join the complimentary DNA nucleotide bases and the single strand DNA by hydrogen bonds and adjacent nucleotides by phosphodiester bonds
What are the advantages of using the ‘gene machine’ ?
quick
any sequence can be made
high accuracy
no introns
what is the purpose of PCR ?
it used to amplify the DNA fragments in a short period of time
What are some genetic engineering procedures that require PCR ?
-DNA sequencing.
- Gene cloning.
- DNA profiling.
- Making artificial genes.
Explain the purpose of PCR
To produce large quantities of ‘cloned’ DNA from a very small sample
Why is DNA (taq) polymerase from bacteria living in hot springs used for PCR ?
it is very stable at high temperatures, does not denature
After 2 cycles of replication 4 copies of double-stranded DNA exist. Calculate how much a DNA sample will have increased after 4 cycles.
8 copies
Explain what the effect would be of having a single molecule of unwanted DNA in the sample prior to PCR
it would be amplified along with the intended DNA sample, contaminating the sample and rendering it as unusable
Suggest possible sources of DNA contamination in preparing a DNA sample
dirty equipment that has DNA molecules left on it from previous treatments
viruses and bacteria in the air
DNA from technician
State precautions that could be taken to reduce the risk of DNA contamination
using disposable equipment
use of sterile procedures
What causes DNA replication to stop in PCR ?
nucleotides and primers used up.
DNA polymerase denatures.
What are the features/advantages of In-vitro cloning ?
PCR copies DNA that has been partly broken down/ doesn’t require living cells
useful in forensic science
Rapid-billions of copies in a short period of time.
PCR is very sensitive. Minute amounts of DNA, such as that contained in a single cell can be copied
DNA embedded in other material can be copied. Useful for analysing DNA in archaeological remains
cloned genes produced in solution. They cannot be used directly to manufacture the protein that they encode.
DNA polymerase will only copy a gene if it is marked at each end by complimentary primers. If we do not know the base sequences at each end of the gene, we cannot make appropriate primers.
Cannot be used to copy genes that have not been studied before.
PCR unreliable if DNA fragments longer than about 1000 base pairs
lack of error-correcting mechanisms, so the error rate is higher.
What are the features/advantages of In vivo cloning ?
partly broken down DNA not copied
Used for introducing genes into other organisms
unlikely to be contaminated
In vivo methods are less sensitive, so large amounts of sample DNA are needed. Less useful in forensic work.
Unless DNA can be isolated from the medium in which it is embedded, it cannot be copied.
cloned genes already inside cells that will manufacture the protein that they encode
once incorporated into its host DNA, the gene will be copied by the host cell
This method can be used to copy genes that have not been studied before.
methods reliably copy genes up to about 2mbp long- precise
cells have mechanisms for correcting any errors that are made when copying genes therefore its accurate.
Explain how a gene machine is used to artificially produce genes
The sequence of amino acids and bases is determined for the required gene.
The desired sequence of nucleotide bases for the gene is inputted into a computer
The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements.
The computer designs a series of small overlapping single strands of nucleotides, called oligonucleotides
The oligonucleotides are linked together- no introns have been copied
The gene is replicated using PCR to make multiple copies.
What are the advantages of using the gene machine to produce DNA fragments ?
Quick
any sequence can be made
high accuracy
no introns
When restriction endonucleases are used to cut the DNA they can leave fragments with two straight edges what are these known as ?
Blunt ends
When restriction endonucleases are used to cut the DNA they can leave fragments with uneven edges. What are these known as ?
Sticky ends
What is a DNA probe ?
