Chapter 6: Biotechnology - Its Tools & Techniques Flashcards
ingredients required for PCR
template DNA strand
free nucleotides
taq DNA polymerase enzyme
primers
buffer solution
what are primers
short, single stranded pieces of chemically synthesised DNA which flank the target region
define buffer solution
liquid into which all the all of the other ingredients are added: prevents sudden pH changes.
where is PCR done?
to do a PCR, mixture is put into a thermal cycler, so it can be heated and cooled in a controlled way. PCR uses variations in temperature to control the replication process
steps of PCR
- Denaturation
- PCR reaction mixture is heated to 95 degrees celsius
- DNA is denatured, i.e. separates to form two single strands - Annealing
- PCR reaction mixture is cooled down to 55 degrees celsius
- allows primers to anneal (=bind) to the template DNA - Extension
- PCR reaction mixture is heated to 72 degrees celsius
- DNA polymerase moves down the template, synthesising new DNA.
does the amount of DNA per PCR cycle quadruple?
In each cycle, the number of DNA molecules doubles.
what does gel electrophoresis do ?
separates ad visualises DNA fragments based on their size
what is DNA charge?
negative due to the phoshpate group
steps of gel electrophoresis
- prepare the set up:
- Load DNA samples and ladder:
- Run the gel
well definitnion
hole with a specific size
Setting up stage of gel electrophoresis
- prepare the set up:
- pour the liquid agarose gel into a mould
- before it sets, insert a well comb.
- once mould has set, place it into a gel box which is set up with a negative electrode on one end, and a positive electrode on the other end. wells are on the side with the negative electrode
- Immerse gel with buffer solution (can conduct electricity)
loading DNA samples and ladder stage of gel electrophoresis
- Load DNA samples and ladder:
- Each DNA sample is transferred into its own well, these are usually amplified using PCR before they are run through the gel, so there are enough to use.
- A DNA ladder is added into one well, this ladder is a mixture of known DNA fragments, each of which has a specific size.
running the gel phase of gel electrophoresis
- Run the gel:
- Turn on the power - a current runs through the gel
due to the negative charge of DNA from its phosphate groups, and the fact that opposites attract, DNA moves through gel.
- DNA migrates through the gel, towards to positive pole.
why can DNA be separated based on size
Gel acts as a matrix. smaller DNA fragments move faster than bigger longer ones do.
what does the electircal current in gel elcotrorei do
provides a force to move the DNA
what does the agarose gel do in gel elelctoroepfero
strucutres (tiny pores) filters DNA by size.
what does the DNA ladder allow us to do?
to infer how many base pairs are in each fragment in the wells. thus, how long each of them are.
visualising the DNA fragments phase of gel electrophoresis
- Visualising the DNA fragments:
- Add a dye to stain the DNA fragments, by either:
a) to liquid agarose, before its poured into the mould
b) to gel, after it has been run.
define band in gel eleoeroeikvl;
each of the lines in the gel. they contain large numbers of DNA fragments of the same size, which have ultimately travelled to the same position in the gel.
Applicaitons of gel electrophoresis.
DNA sequencing
DNA profiling
Recombinant DNA
Describe how a specific gene from extracted DNA could be amplified.
PCR can be used to amplify this specific region of DNA. Complementary primer strands to the start and end of the gene are added. This is followed by PCR cycling (heating and cooling) with polymerase enzymes to replicate the region, amplifying the gene.
A high school biology student wants to run a gel electrophoresis experiment, but he accidentally loads the gel backwards. The DNA is closest to the positive electrode.
Predict what might happen when he turns on the power.
The negatively charged phosphate groups in DNA’s backbone are attracted to the positive electrode. If the gel is loaded backwards with the DNA closest to the positive electrode, it won’t be pulled through the gel and separate, but instead will go backwards out of the gel.
Explain the role of the gel in gel electrophoresis.
The gel is a matrix, meaning it is porous and acts like a filter for DNA. This separates DNA based on size, as shorter DNA fragments can migrate more quickly through the pores, and so will travel further along the gel in a set amount of time.
Applications of DNA sequencing
In medicine, DNA sequencing can be used to determine if a patient is at risk of a genetic disease.
DNA sequencing is a useful tool in scientific research because it can be used to study genomes and the proteins they encode, at a molecular level.
DNA sequencing is used in evolutionary biology to determine inheritance patterns.
The uniqueness of a person’s genetic code means that we can identify them by sequencing their DNA.
defone dna profiling
DNA profiling refers to the process of analysing DNA variations, for the purpose of identification. these variations in DNA are identified via genetic markers, regions of DNA which usually vary between individuals. these are used to make DNA profiles, most common genetic markers are STR.
define STRs
Short tandem repeat is a string of repeating nucleotide units. usually 2-5 bases long. they are non-coding DNA.
steps of DNA profiling
coleklcting DNA sample stage of DNA profiling
- Collect DNA sample:
Obtained from any material that contains cells
e.g. saliva, blood, hair
extracting DNA sample stage of DNA profiling
- extract DNA sample
chemicals (i.e. detergents) are added which break open cells
DNA is separated from the other cell components e.g. proteins
- Amplify STR fragments
involves using PCR to make copies of <13 different STR regions
this is achieved by using primers which flank the region by binding to the DNA on each side of the STR.
- Determine lengths of the STRs
amplified PCR fragments are separated by gel electrophoresis
larger fragments move through the gel slower, compared to smaller fragments. (largest fragments = have most repeats, smallest fragments = least repeats)
- interpreting the electrophoresis gel:
patterns of bands on the electrophoresis gel (i.e., the number of bands and their locations) create a DNA profile
can determine the number of repeats in the STR by referring to the DNA ladder.
steps of making recombinant DNA
isolation -> digestion -> insertion -> ligation
Explain an advantage of using DNA recombinant technology, rather than artificial selection, to produce a herbicide-resistant strain of canola.
Using DNA recombinant technology allows a gene to be taken from unrelated organisms that provide herbicide resistance. Therefore, the chance of finding a suitable gene is greatly improved, compared to having to find a closely related herbicide-resistant plant to breed with.
Using DNA recombinant technology is quick to incorporate the herbicide-resistant trait into the species. Whereas, artificial selection can require numerous generations to obtain pure-breeding herbicide-resistant plants.
Using DNA recombinant technology allows for only the herbicide-resistant trait to be manipulated. Whereas, artificial selection may also lead to changes in other related traits.
Describe how recombinant DNA technology can be used to genetically modify bacteria to produce chymosin.
DNA is extracted from the calves, and the chymosin gene is isolated and removed using restriction enzymes
The bacterial plasmid is cut, using the same restriction enzymes, and the chymosin gene inserted into the gap
Bacteria are transformed, as electroporation or heat can be used to promote uptake of the recombinant plasmid
The transformed bacteria are cultured and multiply, expressing the chymosin gene to produce the chymosin enzyme
Explain how gene A and gene B could be monitored to determine which bacterial hosts have successfully incorporated the desired recombinant DNA.
Genes produce necessary proteins when they are fully intact. If a gene has been ‘broken’ to allow for the insertion of another gene, it will not be functional. To identify which bacteria have been transformed with recombinant plasmids (as opposed to normal plasmids), the bacteria should be grown on nutrient agar plates, which also contain the antibiotic Ampicillin. Both bacteria containing a non-recombinant plasmid will grow because gene A is intact and expresses resistance to Ampicillin. If gene B is intact (non-recombinant) they will also appear yellow in colour. But bacteria containing the desired recombinant plasmid will not be yellow in colour because the desired gene X has been spliced into the middle of gene B which is unable to be expressed to produce the yellow substance.
define biotechnology
using biological processes, systems and organisms for the benefit of humanity.
define transgenic organisms
GMOs which have been modified to contain certain genes from another species.
define transgenics
Transgenics is the artificial introduction of a DNA sequence into an organism from a different species, known as a transgene. This gives the transgenic organism the ability to express the trait encoded by the transgene.
Describe a process used to produce GMOs.
Genetically modified organisms are organisms that have had their genome modified. Transgenic organisms are a type of GMO, in which a transgene is introduced that codes for a desirable trait. A common method for producing transgenic organisms is using bacterial plasmids. This involves introducing the gene of interest into the bacterial plasmid to produce recombinant DNA. Then, certain bacteria can be used that are able to insert their plasmid into the DNA of the target organism, modifying its genome.
Distinguish between genetically modified organisms (GMOs) and transgenic organisms.
Genetically modified organisms are organisms that have been genetically engineered in any way to have modified DNA. Transgenic organisms are organisms that have had a transgene added into their genome. Therefore, transgenic organisms are a type of GMO, however not all GMOs are transgenic organisms.
Propose how the Bt cotton plant was first produced and the need for it.
Bt cotton is a genetically modified organism (GMO), meaning it has had its genome modified. Specifically, it is a transgenic plant that has had a transgene introduced into its genome. This is often done using recombinant DNA with bacterial plasmids as vectors. These are introduced into the genome by bacteria that insert the plasmid into the host cell’s DNA. Unlike regular cotton, Bt cotton will produce a toxin that kills caterpillars that eat the crop. This will increase the productivity and yield, making the farming of the cotton more profitable, decreasing the price of cotton.
DNA Ligase
Catalyses a covalent bond b. [phosphodiester bond] with between nucleotides [1 mark]
enzyme
DNA Polymerase
Attaches free nucleotides [one at a time] to the template strand, [1mark]
according to complementary base pairing rule [1 mark]
enzyme
Primers
Anneals to DNA Strand at a specific point to indicate where the polymerase begins synthesis [1mark]
short fragments of single-stranded DNA or RNA.
Restriction enzyme what are they and what is their function
- Restriction site
- enzyme
Describe the role restriction enzymes play in transferring the genes
- USED TO CUT THE TARGET SEQUENCE FROM THE ENDERBY ISLAND CATTLE DNA,
- USED TO CUT THE BACTERIAL PLASMID (VECTOR)
- THEREFORE, ENDS (STICKY) OF RESTRICTION FRAGMENT AND PLASMID ARE COMPLEMENTARY
- ALLOWING THEM TO BE JOINED BY DNA LIGASE
Name two possible positive outcomes resulting from crop genetic modification.
- INCREASE YIELD
- REDUCE THE NEED TO SPRAY INSECTICIDE
Outline the sequence of events undertaken to produce Bt cotton.
- CUT Bt GENE OUT- USING RESTRICTION ENZYME to create RESTRICTION FRAGMENT [1 mark]
- CUT BACTERIAL PLASMID/AGROBACTERIUM USING THE SAME RESTRICTION ENZYME [1 mark]
- STICKY ENDS OVERLAP AND ARE COMPLEMENTARY TO EACH OTHER BECAUSE THEY HAVE BEEN CUT WITH THE SAME RESTRICTION ENZYME. [1 mark]
- Bond/Join/LIGATE by DNA LIGASE, (phosphodiester bonds) Bt GENE INTO PLASMID/AGROBACTERIUM – RECOMBINANT DNA [1 mark]
- TRANSFORMATION: RECOMBINANT PLASMID/AGROBACTERIUM MIXED WITH BACTERIA, SOME BACTERIA TAKE UP THE RECOMBINANT PLASMID. [1 mark]
- SELECTION: BACTERIA PLACED IN CULTURES CONTAINING AMPICILLIN/ANTIBIOTIC. ONLY TRANSFORMED BACTERIA SURVIVE. [1 mark]
- TRANSFORMED BACTERIA INTRODUCED INTO THE COTTON
The use of genetically modified (GM) crops has been controversial. Describe an example of an issue related to the use of GM crops.
- Roundup ready crops leading to over use of pesticides/ Toxin resistance - eg aphids and mealy bugs
- Contamination of organic crops
- Possible effects on non-target organisms: Bt cotton and beneficial insects, pollen of pest-resistant corn and butterflies, bees (species that feed on Bt cotton or GM corn)
- Outcrossing: migration of genes from GM plants to conventional crops or related wild species
- Superweeds
- Reduction in genetic diversity/monoculture
What is the purpose of PCR and what are the steps involved in this process?
- TO AMPLIFY/INCREASE THE AMOUNT OF DNA [1], doubles with each cycle (1/2 mark)
- STEPS:
1. DENATURATION[1/2] double stranded DNA is heated to 95oC, breaking the H-bonds/denature [1/2], exposing nucleotide bases. (1/2)
2. ANNEALING [1/2] temperature is lowered – 50-60oC, DNA primers anneal at opposite ends/3’ end. (1 mark)
3. EXTENSION [1/2] temperature raised – 72oC, optimum temperature for DNA taq polymerase, synthesis begins (1 mark)
What is the purpose of gel electrophoresis and what are the steps involved in this process?
- TO SEPARATE DNA STRANDS BY LENGTH/SIZE (1 mark)
- STEPS:
o DNA samples and ladder (‘ruler’- standard set of molecule size markers [1/2]) added to wells (in agarose gel) (1)
o Electric current turned on- DNA molecules/fragments move towards the positive (cation) end (1) (DNA is negatively charged due to phosphate group) (1/2)
Smaller fragments move faster and end up further along the gel. (1/2)
o Visualise- fragments are photographed, they appear as bands. Fragments have been tagged with a dye in order to be seen. (1)
o Comparison of samples. (1/2)
