MANIPULATING GENOMES Flashcards
Techniques to study genes and their function
PCR
Gel electrophoresis
Cutting out DNA fragments using restriction enzymes
These techniques are used in DNA profiling, DNA sequencing, genetic engineering and gene therapy
PCR
Makes multiple copies of a DNA fragment
Used to select a fragment of DNA containing part of the DNA that you’re interested in and amplifying it to produce millions of copies in just a few hours
Steps of PCR
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase
The DNA mixture is heated to 95oC to break the hydrogen bonds between 2 strands of DNA, DNA polymerase doesn’t denature even at this high temperature and this is important because it means many cycles of PCR can be carried on without having to use new enzymes each time
Mixture is cooled to between 50oC and 65oC so that primers can bind to strands
The reaction mixture is heated to 72oC so that DNA polymerase can work
DNA polymerase lines up free DNA nucleotides alongside each template strand and complementary base pairing takes place
2 new copies of the fragments of DNA are formed and one cycle of PCR is complete
Cycle starts again with mixture being heated to 95oC and this time all 4 strands are used as templates
Each PCR cycle doubles the amount of DNA
Electrophoresis
Separates DNA fragments by size
Uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size
Steps of electrophoresis
Use of agarose gel that has been poured into a gel tray and left to solidify, a row of wells is created at one end of the gel, the end of the tray with these wells is closest to the negative electrode on the gel box
Add a buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution
Take your fragmented DNA samples and, using a micropipette, add the same volume of loading dye to each (loading dye helps samples sink to the bottom of the wells so they’re easier to see)
Add a set volume of DNA sample to your first well and make sure the top of the micropipette is in the buffer solution and just above the opening of the well
Repeat the process and add the same volume of each of your other DNA samples to the other wells in the gel using a clean pipette each time
Record which DNA sample you added to each well
Put the lid on the gel box and connect the leads from the gel box to the power supply and turn the power supply on and set it to required voltage causing an electrical current to pass through the gel
DNA fragments are negatively charged and move towards the positive electrode at the far end of the gel (anode) small DNA fragments move faster and travel further through the gel so the DNA fragments separate according to size
Let the gel run for 30 minutes and turn off the power supply
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 rinse the gel with water, the bands of different DNA fragments will now be visible, the DNA fragment is measured in bases
What would you have to do first if you use proteins in electrophoresis
Mix it with a chemical that denatures the proteins so they all have the same charge
Steps of cutting out DNA fragments using restriction enzymes
Some sections of DNA have palindromic sequences of nucleotides, these sequences consist of antiparallel base pairs
Restriction enzymes are enzymes that recognise specific palindromic sequences and cut the DNA at these places
Different restriction enzymes cut at different specific recognition sequences because the shape of the recognition sequence is complementary to an enzymes active site
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
The DNA sample is incubated with the specific restriction enzymes which cut the DNA fragment out via a hydrolysis reaction
The cut leaves sticky ends- small tails of unpaired bases at the end of each fragment they can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary base pairs
DNA profiling- electrophoresis
Some of the organisms genome consists of repetitive, non coding base sequences
The number of times these sequences are repeated differs from person to person so the length of these nucleotides differs too
The number of times a sequence is repeated at different, specific loci in a persons genome can be analysed using electrophoresis which creates a DNA profile
The probability of individuals having the same DNA profile is very low because the chance of 2 individuals having the same number of sequence repeats at each DNA locus in DNA is very low
DNA profiling- forensic science
Forensics use DNA profiling to compare the samples of DNA collected from crime scenes to samples of DNA from suspected suspects, linking them to the crime scene
DNA is isolated from all the collected samples
PCR is used to amplify multiple areas containing different sequence repeats
PCR products are run on an electrophoresis gel and the DNA profiles produced are compared to see if there is any match
DNA profiling- medical diagnosis
In medical diagnosis a DNA profile can refer to a unique pattern of several alleles
Can be used to analyse the risk of genetic disorders
Useful when the specific mutation isn’t known or where several mutations could have caused a disorder because it identifies a broader, altered genetic pattern
Genetic engineering
Transformed organism- an organism that has had their DNA altered by genetic engineering
These organisms have recombinant DNA, DNA that is formed by joining together DNA from different sources
Genetic engineering involves extracting a gene from one organism and inserting it into another organism
Genes can be manufactured instead of extracted from an organism
The organism with the inserted gene will produce the protein coded for by that gene
An organism that has been genetically engineered to include a gene from a different species is called a transgenic organism
Process of genetic engineering
DNA fragment containing the gene you want is isolated using a restriction enzyme
DNA fragment is inserted into vector DNA- a vector is something that is used to transfer DNA into a cell (plasmids of bacteriophages)
The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene, the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
The vector DNA and DNA fragment are mixed together using DNA ligase which joins up the sugar-phosphate backbones of the 2 bits (ligation)
The new combination of bases in DNA is called recombinant DNA
The vector with the recombinant DNA is used to transfer the gene into the bacterial cell
If a plasmid vector, the bacterial cells have to be persuaded to take in the plasmid vector and it’s DNA
With a bacteriophage vector, the bacteriophage will infect the bacterium by injecting its DNA into it, the phage DNA then integrates into the bacterial DNA
Cells that take up vectors containing the desired gene are genetically engineered and so are called transformed
Examples of genetic engineering
Insect resistance in plants
Producing drugs from animals
Pathogens for research
Genetically engineered organisms ‘owned’ by big companies
Insect resistance in plants
Soy beans are an important food source across the world but yields are reduced by insect pests
Scientists have genetically modified plants to include a gene originally found in the bacteria ‘Bascillus thuringiensus (Bt)’ which codes for a protein that is toxic to some insects that feed on soy bean plants
To genetically modify a soybean plant a desired gene is isolated from Bt using a restriction enzyme and inserted into a plasmid taken from the bacterium Agronacterium tumefaciens, the plasmid is put back into the bacterium and the soybean cells are deliberately infected with the transformed bacteria, the desired gene gets inserted back into the soybean plants DNA creating a genetically modified plant
GM insect resistance in plants positive ethical issues
Reduces the amount of chemical pesticides that farmers use on their crops which could harm the environment
