3.5 Genetic Modification and Biotechnology Flashcards
Method of DNA Extraction
- isolate the DNA from the rest of the cell - by mechanically breaking cells open, then using detergents and enzymes to break down cell walls and membranes
- remove unwanted cell debris - by either filtering the extract or centrifuging the mixture
- remove the unwanted proteins - by adding protease enzyme that destroys proteins, then some phenol will be added to destroy all of it
- precipitate out the DNA - by pouring a layer of ice-cool ethanol over the surface of the filtrate
Restriction Enzymes (endonuclease)
are enzymes that cut DNA at specific sequences of nucleotides (recognition site)
bacteria, when attacked by viruses have a defence mechanism where they chop up the invading viral DNA using the enzymes
this was harnessed by scientists to use as DNA scissors
named according to the bacterial species and then numbered
Blunt end
the DNA is cut straight across by an endonuclease
Sticky End
also called palindromic cuts as they make staggered cuts, giving sequences in which both strands read the same in the 5’ to 3’ direction
most commonly used
these ends can be annealed (glued to some DNA from a different source that has been cut by the same restriction enzyme)
Ligase Enzymes
combining the sticky ends of two different DNA strands is only temporary due to only a few H bonds holding ends together
the joins can be made permanent using the ligase enzyme as they catalyse the formation of bonds between the phosphates and the sugars on the side of the DNA ladder during DNA replication and repair
Polymerase Chain Reaction (PCR)
can be used to amplify small amounts of DNA (i.e. produce multiple copies of DNA rapidly)
method:
1. DNA sample is placed in a PCR tube along with a mix of buffer solution, primers, free nucleotides and Taq polymerase
2. Then, at high temperatures (90) the DNA is split
3. the temperature is then dropped to 55 so that specific primers are able to bind to the DNA
4. the temperature is then raised again to 72 so that a new strand can be synthesised by nucleotide units
Applications of PCR
- used by police in forensics if only a small amount of tissue is found
- anthropologists and archeologists use it to check ancient fossils
- used to see if a person has a specific gene by careful choice of primer
- used to identify viral genes
- rapidly able to identify any prenatal genetic disorder from a few fetal cells
Gel Electrophoresis
is used to separate proteins or fragments of DNA according to size
depends on the facts:
- when restriction enzymes have cut DNA, the fragments will be of different lengths
- the DNA is strongly negative due to the phosphates in the nucleotides
method:
1. an agarose gel is made with wells in it
2. the whole box is flooded with a buffer solution which will allow electricity to pass through
3. the prepared DNA is carefully introduced into the small wells
4. the electrodes are turned on, at each end of the box, which the positive electrode (anode) furthest away
5. the DNA will move through the gel with the smallest pieces moving the fastest towards the positive electrode
6. the rate at which the fragments move is inversely proportional to their length (so their exact length can be calculated)
DNA Profiling
involves the comparison of DNA
method:
1. DNA is extracted from the sample (PCR may be needed to amplify sample)
2. the DNA sample is treated with the restriction enzyme HinfI which cuts the DNA in several places
3. Gel electrophoresis allows different lengths of DNA to be separated
4. treatment of the gel with radioactive probes complementary to the part of the sequence of the VNTR or STR regions to be analysed
5. autoradiograph is produced
What is a probe?
a single strand of DNA that will stick to a gene of interest
as it is complementary to the gene of interest
Applications of DNA Profiling
- forensics, to match suspect with scene evidence
- paternity testing
- identifying presence of a particular gene
- genetic relatedness of different organisms
- breeding programmes
Methods of Genetic Modification
- adding a foreign gene: which will enable the GMO to carry out a new genetic programme, (called transgenic organisms)
- altering an existing gene: altered to make it be expressed at a higher level or in a different way (used for gene therapy)
- deleting or turning off a gene: an existing gene may be deleted or deactivated to prevent the expression of a trait
How is Genetic Modification possible?
because the genetic code is universal
it involves the gene transfer between species
Genetically Modified Bacteria Method (using production of human insulin)
bacteria contains additional circular DNA called plasmids that can be easily removed, modified and inserted
method:
1. isolate a gene of interest using restriction enzymes (e.g. insulin gene)
2. remove plasmids from bacteria
3. cut plasmid with the same restriction enzyme
4. stick the cut plasmid and gene of interest together using ligase
5. new recombinant plasmid is inserted back into bacteria
6. bacteria reproduce asexually and express the insulin gene
Possible benefits of GMO Crops
- the nutritional value of food could be improved
- crops can be produced that lack common allergens
- crops can be grown in arid conditions that produce a high yield
- GM crops can produce herbicides to kill pests
- improve food supply in poorer countries
- reduces economic costs
