Manipulating Genomes Flashcards
What are the three techniques used to study genes
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Cutting out DNA fragments using restriction enzymes
What can the polymerase chain reaction be used for
To select a fragment of DNA and amplify it to produce millions of copies in a few hours
What are the three stages of PCR
Separation, annealing of primers, synthesis of DNA
What happens during the separation stage of pcr
A reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is set up. It is heated to 95 degrees Celsius to break the hydrogen bonds between DNA strands.
What happens to DNA polymerase during the separation stage of PCR
It does not denature even at this high temperature so many cycles of PCR can be carried out with the same enzymes
What happens during the annealing of primers stage in PCR
The mixture is cooled to 55 degrees Celsius so that the primers can anneal (bind) to the strands.
What happens during the DNA synthesis stage of PCR
The reaction mixture is heated to 72 degrees Celsius so that DNA polymerase can work. It lines up free DNA nucleotides alongside each template strand. Complementary strands can then be formed.
How many new copies of the fragment of DNA are formed in one cycle of PCR
2
What are primers
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
What is DNA polymerase
An enzyme that creates new DNA strands
What is electrophoresis
A procedure that uses an electrical current to separate out DNA fragments, RNA fragments or proteins dependent on their size.
What is the set up that allows electrophoresis to occur
- Performed using agarose gel in a gel tray.
- This is then placed into a gel box or tank.
- At the negative end (cathode), the gel has wells. Using a micropipette, the fragmented DNA samples with loading dye should be added to the wells.
- There should be a buffer solution added to the reservoirs at the side of the gel box so the surface of the gel becomes covered.
How to carry out electrophoresis
- Put the lid on the gel box and connect the leads from gel box to the power supply.
- Turn on the power supply and set to required voltage (100 V).
- DNA fragments are negatively charged and therefore will move through the gel towards the positive electrode at the far end of the gel
- Let the gel run for around 30 mins (or until dye is about 2cm from end of gel) and turn off power supply
Which DNA fragments move the quickest in electrophoresis
Small DNA fragments will travel faster and further as they will separate according to size
What to do once electrophoresis has finished
Remove the gel tray from the gel box and tip off any excess buffer solution.
Wearing gloves stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water. The bands will now be visible
What must you do before carrying out electrophoresis on proteins and why
They may be positively or negatively charged so they are mixed with a chemical that denature the protein so they all have the same charge.
What is the use of electrophoresis in proteins
To identify the proteins present in urine or blood samples - help diagnose disease
What are restriction enzymes
Enzymes that recognise specific palindromic or recognition sequences and cut the DNA at these places
What are palindromic or recognition sequences
A palindromic sequence is a nucleic acid sequence in a double-stranded DNA or RNA molecule whereby reading in a certain direction on one strand is identical to the sequence in the same direction on the complementary strand.
Why do different restriction enzymes cut at different recognition sequences
Because the shape of the recognition sequence is complementary to an enzymes active site
What can occur if recognition sequences are present at either side of the DNA fragment you want
You can use restriction enzymes to separate it from the rest of the DNA
What happens to the DNA sample with specific restriction enzymes
It is incubated and the restriction enzymes cuts the DNA fragment out via a hydrolysis reaction
What is sometimes left after using restriction enzymes to cut DNA fragments and what can it be used for
Sticky ends (small tails of unpaired bases at each end of the fragment. They can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
Why is electrophoresis used
To produce DNA profiles as:
The number of times a persons non-coding sections are are repeated differs from person to person, so the length of nucleotides differ too
The number of times a sequence is repeated at different specific places in a persons genome to create a DNA profile
The probability of two individuals having the same DNA profile is very low
How can DNA profiling be used in forensic science
To compare samples of DNA collected from crime scenes to samples of DNA from possible suspects, to link them to the crime scenes
Method forensic scientists use when trying to link the person to the crime scene
1) DNA is isolated from all collected samples
2) PCR is used to amplify multiple areas containing different sequence repeats - primers are used to bind to either side of these repeats and so the whole repeat is amplified
3) The PCR prodcuts are run on electrophoresis gel and the DNA profiles produced are compared to see if any match is there
How can DNA profiling be used in medical diagnosis
It refers to a unique patter of several alleles and can be used to analyse the risk of genetic disorders. It is useful when the specific mutation isn’t known or where several mutations could have caused the disorder as it identifies a broader, altered genetic pattern
What is genetic engineering
The manipulation of an organism’s DNA
What are organisms called that have had their DNA altered by genetic engineering
Transformed organisms/genetically modified organisms
What is recombinant DNA and where is it found
DNA formed by joining together DNA from different sources
Found in transformed organisms
What a transgenic organism
An organism that has been genetically engineered to include a gene from a different species
What are the five stages of genetic engineering
Isolation, insertion, transformation, identification, growth
What are the two ways that isolation can occur
Reverse transcription
Restriction endonucleases
How does reverse transcription result in isolation of a gene during genetic engineering
Reverse transcriptase makes DNA from an RNA template - opposite to transcription
This is by joining DNA nucleotides with complementary bases to the mRNA sequence.
Single stranded cDNA is made, and to make this double stranded DNA polymerase is used
How are restriction endonucleases used to isolate the desired gene
They cut the viral DNA at specific sites as gene probes can locate the desired gene on a DNA fragment.
They have highly specific active sites so be able to cut the recognition sites which are usually palindromic.
When using restriction endonucleases what is the difference with cutting blunt or sticky ends
Blunt ends = restriction enzymes cut straight across both chains forming blunt ends
Sticky ends = restriction enzymes mostly make a staggered cut. They have complementary single stranded DNA which will join with another sticky end if it has been cut with the same restriction enzyme
What happens during the insertion stage of genetic engineering
1) The DNA fragment is inserted into vector DNA
2) The vector DNA is cut open using the same restriction enzyme that isolated the fragment of desired DNA, so that the sticky ends are complementary
3) The vector DNA and fragment DNA are mixed together with DNA ligase forming recombinant DNA
What are vectors in genetic engineering
Something that’s used to transfer DNA into a cell, they can be plasmids or fungi
What does DNA ligase do and what is the process called
Joins the sugar-phosphate backbones of the two fragments of DNA
Called ligation
What happens during transformation of genetic engineering
The vector with the recombinant DNA is used to transfer the gene into the bacterial cells.
If the cells take up the vectors gene they are transformed.
How to ensure the gene is taken up to create the required protein in transformation
The cell membrane must be more permeable by electroporation and heat shocking
What is electroporation
When an electrical field is created in the mixture which increases the permeability of the bacterial cell membranes
What are the two ways to be able to tell if a host cell has take up the plasmid
Fluorescent markers
Antibiotic - resistance marker genes
How are fluorescent markers used in identification
The green fluorescent protein has been incorporated into a plasmid and if the DNA fragment has been inserted into the GFP gene the bacterial will not glow and be identified.
If it has not been inserted then the bacteria will glow
How are antibiotic resistance markers used in identification
By growing the bacteria on a medium containing tetracycline antibiotic.
The antibiotic resistant gene is found in the plasmid only and therefore the bacteria that survive contain the plasmid
What happens in the growth stage of genetic engineering
A fermenter is used to grow multiple copies of the host cell that have been identified to contain the recombinant plasmid
What ways can genetic engineering be useful
Create insect-resistance in plants
Produce drugs from animals
Research into pathogens to find treatments to diseases
What are the positive ethical issues concerning GM plants
Reduce the amount of chemical
What are the advantages of GM farmed animals
- Improves quality
- Improves quantity
- Improves productivity
What is pharming
The production of pharmaceuticals and human medicines by inserting human genes into other animals, allowing for the extraction of pharmaceutical proteins from GM animal’s milk or blood at high yields
Advantages of pharming
- Enables mass production of rare treatments
- Makes drugs more accessible
Disadvantages of pharming
- Raises animal welfare concerns
- Can lead to animals being viewed solely as commodities
What is germ line therapy
Inserting a healthy allele into the germ cells or into a very early embryo
What is somatic cell gene therapy
Replacing a faulty gene with a healthy allele in affected somatic cells
Is germ line therapy good or not
It is currently illegal as ensures the offspring inherits the corrected gene permanently
Does somatic cell gene therapy prevent the next generation from getting the disease
No as it does not affect the sperm and egg cells. It has to be repeated many times as the effects do not last very long.
What are the three ways of completing somatic cell therapy
Repair the gene responsible for the disease
Replace the defective gene with a normal one
Add a normal gene but leave the defective one in
