Manipulating Genomes (6) Flashcards
What are some techniques used in DNA profiling, DNA sequencing, genetic engineering, and gene therapies?
- PCR
- Gel electrophoresis
- cutting out DNA fragments using restriction enzymes
What is the premise of the polymerase chain reaction (PCR)?
It can be used to select a fragment of DNA and amplify it to produce millions of copies in just a few hours. PCR has several stages and is repeated over and over to make lots of copies
What happens during the first step of PCR (denaturing)?
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers, and DNA polymerase.
The DNA mixture is then heated up to 95 degrees to break the hydrogen bonds between the two strands of DNA. DNA polymerase doesn’t denature at this high temperature so many cycles can happen without needing to replace the enzyme
What are primers?
they are short pieces of DNA that are complementary to the bases at the start of the fragment that you want
What happens during the second step of PCR (annealing)?
The mixture is then cooled to around 65 degrees so the primers can bind to the strands.
The reaction is then heated to 72 degrees so DNA polymerase can work
The DNA polymerase lines up free DNA nucleotides alongside each template strand, complementary base pairing means new complementary strands are formed
What happens during the third step in PCR (Cycling again)?
Two new copies of the fragment of DNA are formed and the cycle is completed
The cycle starts again by heating to 95 degrees and all the 4 new strands are used as templates- resulting in exponential growth of DNA molecules
What is electrophoresis?
Electrophoresis is a process that uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size.
How would you carry out electrophoresis (prep)?
- make up a gel by pouring agarose gel into a gel tray and leave to solidify, make sure to use a comb to create a row of wells on one side
- to perform make sure you put the gel tray into the tank with the wells closer to the negative side
- add buffer solution to the reservoirs at the sides of the gel box so the surface of the gel becomes covered in the buffer solution
How would you carry out electrophoresis (DNA loading)?
- use a micropipette to add the same volume of loading dye to each of your fragmented DNA samples
- add a set volume of each sample to each well making sure the pipette isn’t pushing through the gel
- make sure to use a clean micropipette each time
- make sure to record which DNA sample you have added to each well
How would you carry out gel electrophoresis (practical)?
- put the lid on the gel box and connect to the power supply
- set the power supply to the correct voltage
As they are negatively charged, DNA fragments will move towards the positive electrode. smaller fragments will move faster and travel further in time - let the gel run for about 30 mins
- remove and tip off any excess buffer solution
- Stain the DNA fragments by covering the surface of the gel with a stainding solution and then using the gel with water. The bands of the different DNA fragments will now be visible.
How can gel electrophoresis be slightly modified for proteins?
before proteins undergo electrophoresis they must be first denatured so they all have the same charge by a chemical .
What are palindromic sequences of nucleotides?
These sequences consist of antiparallel base pairs
What are restriction enzymes?
They are enzymes that recognise specific palindromic sequences known as recognition sequences and cut the DNA at these places
Why are there many types of restriction enzymes?
Different restriction enzymes cut at different specific recognition sequences because the shape of the recognition sequence is complementary to an enzyme’s active site.
What are sticky ends?
Sticky ends are the small tails of unpaired bases at each end of the fragment. Sticky ends ca ne used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
How is electrophoresis used to produce DNA profiles?
- some of the genomes consists of repetitive, noncoding base sequences that can repeat over and over
- the number of times these are repeated varies between people so the length of these sequences in nucleotides differs too
-the number of times a specific sequence is repeated at specific places can be analyzed using electrophoresis
Why is the probability of two individuals having the same DNA profile low?
- the probability of two individuals having the same DNA profile is very low because the chance of two individuals having the same number of sequences repeats at each locus in DNA is very low
How can DNA profiling be used in forensic science?
- DNA is isolated from the sample
- PCR is used to amplify the DNA sample
- the PCR products are run on an electrophoresis gel and are compared to see if there are any matches/similarities
- if the samples match it links a person to a crime scene
How can DNA profiling be used in medical diagnosis?
- a DNA profile can refer to a unique pattern of several alleles
- it can be used to analyze the risk of genetic disorders. It is useful when the specific mutation isn’t known or where several mutations could have caused the disorder because it identifies a broader, altered genetic pattern.
What are transformed organisms?
They are organisms that have had their DNA altered by genetic engineering
What is recombinant DNA?
This is DNA formed by joining together DNA from different sources
What is the basis of genetic engineering?
- it usually contains extracting a gene from one organism and inserting it into another organism
- genes can also be manufactured instead of extracted from an organism
- the organism with an inserted gene will then produce the protein coded for by that gene
What is a transgenic organism?
An organism that has been genetically engineered to include a gene from a different species is sometimes called a transgenic organism
How do you remove a desired gene from DNA?
Restriction enzymes
How is the DNA fragment inserted into a vector? (genetic engineering)
- The DNA fragment is inserted into vector DNA. Example vectors can be plasmids and bacteriophages
- The vector DNA is cut open using the same restriction enzyme used to cut the desired gene so the sticky ends are complementary
- the vector DNA and DNA fragment are mixed together with DNA ligase.
- The new combination of bases in DNA is called recombinant DNA
What does DNA ligase do?
DNA ligase joins up the sugar-phosphate backbones of the two bits. This process is called ligation
How does the vector transfer the gene into the bacteria?
- the vector with the recombinant DNA is used to transfer the gene into the bacterial cells
- if the vector used is a plasmid it needs to be persuaded to be taken in by the bacterial cells
- with a bacteriophage vector the bacteriophage will infect the bacteria by injecting its DNA into it. The phage DNA then integrates into the bacterial DNA
- Cells that take up the vectors containing the desired gene are genetically engineered and are called transformed.
How would you persuade the bacterial cell to take in the plasmid vector?
- a suspension of the bacterial cells is mixed with the plasmid vector and placed in a machine called an electroporator
- the machine is turned on and an electrical field is created in the mixture, which increases the permeability of the bacterial cell membranes and allows them to take in the plasmids
- this technique is called electroportation
How can you create insect resistance in plants?
- produce genetically modified plants with a gene found in a useful bacteria that codes for a protein that is toxic to some of the insects that feed on the plant
- the desired gene can be isolated using restriction enzymes and inserted into a bacterial plasmid. The plasmid is put back into the bacteria and then the plant is infected using this bacteria
- the desired gene then gets inserted into the plant cells’ DNA creating a genetically modified plant
What are some of the positive ethical issued in creating GM plants?
They will reduce the amount of chemical pesticides that farmers use on their crops which can harm the environment
What are some of the negative ethical issues in creating GM plants?
- may encourage monoculture which decreases biodiversity and makes the crops susceptible to disease
What are some of the positive ethical issues with pharming?
Drugs made this way can be made in large quantities compared to other methods of production. This can make them more available to more people
What are some of the negative ethical issues with pharming?
There is concern that manipulating an animals genes could cause harmful side effects for the animal and that using an animal in this way is enforcing the idea that animals are less than humans and can be objectified.
How is the drug ATryn produced in pharming?
- Hereditary antithrombin deficiency is a disorder that makes blood clots more likely to form in the body. The risk of developing blood clots in people with this disorder can be reduced with infusions of the protein antithrombin
- scientists have developed a way to produce high yields of this drug using goats
- initially, fragments of DNA that code for production of human antithrombin in the mammary glands are injected into the goat embryo
- the embryo is then implanted into a female goat, and when the offspring is born it is tested to see if it produces the antithrombin protein. If it does, selective breeding is used to produce a herd of goats that produce antithrombin in their milk
- the protein is extracted from the milk and used to produce a drug that can be given to people with hereditary antithrombin deficiency.
How can genetic engineering be carried out on pathogens for research? (example)
Scientists found that some tumor cells have receptors on their membranes for the poliovirus so the poliovirus will recognize and attack them. By genetically engineering the poliovirus to inactivate the genes that cause poliomyelitis, scientists can use it to attack and kill cancer cells without causing disease. This may lead to a treatment for cancer.
What are the negative issues related to the genetic engineering of pathogens?
- the scientists researching the pathogens could become infected with the live pathogen and potentially cause a mass outbreak of disease
- the genetically modified version of a pathogen could revert back to its original form and cause an outbreak of disease
- in the wrong hands knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents for warfare.
What are the positive ethical issues with ownership in relation to GM organisms?
It means that the owner of the patent will get money generated from selling the product. This encourages scientists to compete to be the first to come up with a new beneficial genetic engineering idea, so we get genetically engineered products faster.
What are the negative ethical issues with ownership in relation to GM organisms?
Farmers in poorer countries may not be able to afford patented genetically modified seeds. Even if they can afford seeds for one year, some patents mean that they are non legally allowed to plant and grow any of the seeds from that crop without paying again. Many people think that this is unfair and that the big companies that own these patents should relax the rules to help farmers in poorer countries.
What is gene therapy?
gene therapy involves altering alleles inside cells to cure genetic disorders
How does gene therapy work for a genetic disorder caused by a dominant allele?
You can silence the dominant allele e.g by adding a section of DNA into the middle of the gene so it no longer works
How does gene therapy work for a genetic disorder that is caused by two recessive alleles?
You can add a working dominant allele to make up for them
How do you insert the new DNA inside the cell in gene therapy?
It is inserted into the cell using vectors:
- viruses
- plasmids
- liposomes
What is somatic gene cell therapy?
This involves alternating the alleles in body cells, particularly the cells that are most affected by the disorder. For example, cystic fibrosis is a genetic disorder that is very damaging to the respiratory system so somatic therapy for CF targets the epithelial cells lining the lungs. Somatic therapy doesn’t affect the individual’s sex cells though so any offspring could inherit the disease.
What is germ-line gene cell therapy?
This involves altering the alleles in the sex cells. This means that every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t inherit the disease. FErm lin therapy in humans is currently illegal though.
What are the positive ethical issues around gene cell therapy?
- it could prolong the lives of people with genetic disorders
- it could give people with genetic disorders a better quality of life
- carriers of genetic disorders might be able to conceive a baby without the risk of cancer
- it could decrease the number of people who suffer from genetic disorders
What are the negative ethical issues around gene cell therapy?
- The technology could potentially be used in ways other than for medical treatment, such as for treating the cosmetic effects of aging
- there is the potential to do more harm than good by using the technology
- there is concern that gene therapy is expensive- some people believe that health service resources could be better spent on other treatments that have passed clinical trials
What are some of the disadvantages of gene therapy?
- effects of the treatment could be short-lived
- the patient might have to undergo multiple treatments
- it might be difficult to get the allele into specific body cells
- the body could identify vectors as foreign bodies and start an immune response against them
- an allele could be inserted into the wrong place in the DNA, possibly causing more problems like cancer
- An inserted allele could get overexpressed, producing too much of the missing protein
What is needed in the chain termination method of sequencing DNA?
- The DNA to sequence (single-stranded)
- lots of primer DNA
- DNA polymerase
- free nucleotides
- fluorescently labeled termination nucleotides
What are the steps in the chain termination method of sequencing DNA?
- each of the four tubes with different fluorescently labelled termination nucleotides undergoes PCR
- the strands formed are different lengths because each one terminates at a different point depending on where the modified nucleotide was added.
- The DNA fragments in each tube are separated by electrophoresis and visualised under UV light
- the complementary base sequence can be read from the gel with the first base at the bottom of the gel so read from the bottom up.
What is the limitation of the chain termination method of sequencing DNA?
It only works on fragments up to about 750 bp long so to do longer samples you need to cut them up and then reassemble them later
How do you use gene sequencing techniques to be used to sequence whole genomes?
- cut the genome into smaller pieces using restriction enzymes
- fragments are inserted into bacterial artificial chromosomes/man-made plasmids
- they are then inserted into bacteria which divide and produce a complete genomic library
- DNA is extracted from each colony and cut up using restriction enzymes to produce overlapping pieces of DNA
- each piece is sequenced using hte chain termination method, and the pieces are put back to give the full sequence from that BSAC
- finally, all the DNA fragments from all the BACs are put back in order by computers to complete the entire genome
How has the chain termination technique for DNA sequencing improved?
- it has been automated and is faster
- a machine reads the sequence for you
What is high throughput sequencing?
They are techniques that can sequence a lot faster than original methods at a fraction of the cost. For example, the chain termination technique has been made high throughput by new technology allowing up to 384 sequences to be run in parallel
What happens in pyrosequencing?
In pyrosequencing, a section of DNA is cut into fragments, split into single strands, and then a strand from each fragment is attached to a small bead. PCR is used to amplify the DNA fragments on each bead, then each bead is put into a separate well. NExt, free nucleotides added to the well attach to the DNA stands via complementary base pairing. The well also contains specific enzymes, which cause light to be emitted when bases are added to the DNA strand. Computers analyze the occurrence and intensities of the light emitted in the different wells and process this information to interpret the DNA sequence. This technique can sequence around 400 million bases in ten hours.
How does sequencing genes link to synthetic biology?
Amino acids are coded by a triplet of base pairs. This means that by sequencing a gene, the sequence of amino acids that the gene codes for and so the primary structure of a polypeptide can be predicted which enables us to create biological molecules.
What are the important parts of synthetic biology?
- building biological systems from artificially made molecules to see whether they work in the way that we think they do
- redesigning biological systems to perform better and include new molecules
- designing new biological systems and molecules that don’t exist in the natural world, but could be useful to humans.
What is computational biology?
This is using computers to study biology
What is bioinformatics?
This is developing and using computer software that can analyze, organise and store biological data
How can sequencing genes be used to study genotype-phenotype relationships?
- it can be useful to predict an organism’s phenotype from its genotype
- For example, Marfan syndrome is a genetic disorder caused by a mutation of a gene. The position and nature of the mutation on the gene affect what symptoms a person with Marfan syndrome will experience. Scientists have sequenced the gene of many people with Marfan syndrome and documented this along with details of their phenotype. Bioinformatics has allowed scientists to compare all the data and identify genotype-phenotype correlations- this could help in the treatment of Marfan syndrome by using gene sequencing to predict what health problems the person is likely to face.
How can sequencing genes be used in epidemiological studies?
Epidemiology is the study of health and disease within a population- it considers the distribution of a disease, its causes, and its effects. Some gene mutations have been linked to a greater risk of disease. Computerized comparisons between the genomes of people who have a disease and those that are done can be used to detect particular mutations that could be responsible for the increased risk of disease.
How can sequencing genes be used to help understand evolutionary relationships?
- All organisms evolved from shared common ancestors. Closely related species evolved away from each other more recently and so share more DNA. Whole genomes of different species can be sequenced and then analyzed using computer software to tell us how closely related the species are.
- Comparing the genomes of members of the same species can also tell us about evolutionary relationships. For example, when different groups of early humans separated and moved to different parts of the world, their genomes changed in slightly different ways. By using computers to compare the genomes of people from different parts of the world, it’s possible to build up a picture of early human migration.
