M6 S4 : Manipulating genomes Flashcards
What are the different techniques used to study genes and their function (the ones I need to know for me exams) ?
- The polymerase chain reaction (PCR).
- Cutting out DNA fragments using restriction enzymes.
- Gel electrophoresis.
What is polymerase chain reaction?
PCR can be used to select a fragment of DNA (containing the gene or bit of DNA you’re interested in) and amplify (increase) it to produce millions of copies in just a few hours.
What are the stages of PCR?
- A reaction mixture is set up containing : the DNA sample, free nucleotides, primers and DNA polymerase.
- The DNA mixture heated at 95 degrees Celsius to break the hydrogen bonds between 2 strands of DNA. DNA polymerase doesn’t denature at this temp, so many cycles of PCR can be carried out , without the need of a new enzyme. Mixture cooled to 55-65 degrees Celsius so primers can bind (anneal) to the strands.
- Temp of reaction mixture increases to 72 degrees Celsius, so DNA polymerase can work. DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairing means new complementary strands are formed.
- 2 new copies of the DNA fragment is formed in one cycle of PCR. The cycle starts again, heated to 95 degrees Celsius and the 4 strands( 2 new and 2 original) used as a template. Each PCR cycle doubles the amount of DNA
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.
Why is DNA polymerase said to a thermostable enzyme?
It doesn’t denature at high temperatures.
What are restriction enzymes also known as?
Restriction endonucleases.
How can restriction enzymes be used to get a DNA fragment from an organism’s DNA?
Restriction enzymes recognise specific palindromic sequences (recognition sequences) and cut (digest) 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 on either side of DNA fragment I want, restriction enzymes can separate it from rest of DNA. The DNA sample is incubated with specific restriction enzyme, which cuts the DNA fragment via a hydrolysis reaction. This can sometimes leave sticky ends. Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
What are palindromic sequences of nucleotides?
These sequences consist of antiparallel base pairs that read the same, but in opposite directions.
What are recognition sequences?
Specific palindromic sequence.
What are sticky ends?
Small tails of unpaired bases at each end of the fragment.
What is electrophoresis?
Electrophoresis is a procedure where an electrical current is used to separate out DNA fragments, RNA fragments or proteins depending on their size.
What are the stages of electrophoresis (PAG 6)?
- It is commonly performed using agarose gel poured into a gel tray, then left to solidify. A row of wells are created on one end of the gel. Put the gel tray into the gel box (or tank). Make sure the end of gel box with wells is closest to the negative electrode on gel box. Then add buffer solution to reservoirs at either side of the gel box, so the surface of gel becomes covered.
- Using a micropipette, add same volume of loading dye to each well. Loading dye helps the sample to sink to the bottom of each well, so easier to use. Next, add a set volume (like 10 micrometres) of a DNA sample to first well. Make sure the tip of the micropipette is in the buffer solution and just above the opening of the well. Don’t stick the tip of micropipette too far into the well, or you could pierce the bottom. Repeat this with the other wells, using a clean micropipette tip each time. Record which DNA samples you have added to each well.
- Put a lid on the gel box and connect the leads from gel box to power supply. Turn the power to the required voltage (like 100V). This causes an electrical current to be passed through the gel. As DNA fragments are negatively charged, the will through the gel towards the positive electrode at far end of gel (anode). Smaller DNA fragments move faster and travel further through the gel, so the DNA fragments will separate out according to their size. Let gel run for 30 mins (or until dye is about 2 cm from end of gel, then turn off power. Remove gel tray from gel box and tip off any excess buffer solution. Wearing gloves, stain the surface of the gel box by covering surface of gel with a staining solution, then rinse the gel in water. This will make the different bands (line) of DNA visible.
What is the charge of a DNA fragment?
Negatively charged.
How can electrophoresis be carried out with RNA fragments and proteins?
Electrophoresis can be carried out with RNA fragments by following the same basic method as for DNA fragments.
Proteins can be either positively or negatively charged, so before they undergo electrophoresis, they are mixed with a chemical that denatures the proteins, so they all have the same charge. This has many uses, like identifying proteins found in urine or blood samples therefore it can help to diagnose diseases.
What are DNA profiles?
Some of an organism’s genome (all genetic material in an organism) consists of repetitive, non-coding base sequences (sequences that don’t code for proteins and repeat over and over, sometimes over thousands of times). The number of times these non-coding sequences repeat differs from person to person, so length of these sequences in nucleotides differ too.
The number of times a sequence is repeated at different, specific places (loci) in a person’s genome (and so the number of nucleotides there) can be analysed using electrophoresis. This creates a DNA profile. The probability of 2 people having the same DNA profile is very low because the chance of 2 individuals having the same number of sequence repeats at each locus in DNA is very low.
How are DNA profiles used in forensic science?
Forensic scientists use DNA profiling to compare samples of DNA collected from crime scenes (e.g. DNA from : blood, semen, skin cells, saliva and hair) to samples of DNA from possible suspects, to link them to crime scenes.
The DNA is isolated from collected samples ( crime scene and suspects). 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. The PCR product is run on an electrophoresis gel and DNA profiles are produced. These are compared to see if there is any match. If they have the same pattern of bands on the gel, then it links that person to the crime scene.
How is DNA profiling used in medical diagnosis?
A DNA profile can refer to a unique pattern of several alleles. It 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, because it identifies a broader, altered genetic pattern.
What is genetic engineering?
The manipulation of an organism’s DNA. Genetic engineering involves extracting a gene from one organism, then inserting it into another organism (often one that is a different species). Genes can also be manufactured (e.g. by PCR) instead of extracted from an organism. The organism with the inserted gene will then produce the protein coded for by that gene.
What are transformed organisms?
Organisms that had their DNA altered by genetic engineering. These organisms have recombined DNA (DNA formed by joining together DNA from different sources).
What is a transgenic organism?
An organism that has been genetically engineered to include a gene from a different species.
What is recombinant DNA?
Formed by joining together DNA from different sources.
What are the names of the 3 steps in genetic engineering?
- Obtaining DNA containing the desired gene.
- Making recombinant DNA.
- Transforming cells.
In genetic engineering, explain how DNA containing the desired gene is obtained?
To get hold of the DNA fragment that contains the desired gene (the gene I am interested in) the fragment can be isolated from another organism using restriction enzymes.
In genetic engineering, what is the process of making recombinant DNA?
This involves inserting the DNA fragment into a vector. The vector DNA is isolated, then restriction enzymes and DNA ligase (an enzyme) are used to stick the DNA fragments and vector together :
- The vector DNA is isolated.
- The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene. This means the sticky ends of vector DNA are complementary to sticky ends of DNA fragment.
- The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sugar-phosphate backbone of 2 bits of DNA. This process is called ligation.
- The new combination of DNA (vector DNA + DNA fragment) is called recombinant DNA.
What is a vector?
Something that is used to transfer DNA into a cell. Vectors can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).
In genetic engineering, what is the process of transforming cells?
The vector with the recombinant DNA is used to transfer the gene into bacterial cells (called host cells). If a plasmid vector is used, the host cells have to be persuaded to take in the plasmid vector and its DNA. Exa : Some bacteria cells are mixed with plasmid vector and placed in a machine called an electroporator. Machine switched on and electric field is created in the mixture. This increases the permeability of the bacterial cell membrane, allowing them to take in the plasmids. This process is called electroporation.
With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The Phage DNA (with desired genes on it) then integrates into the bacterial cell. Cells that take up the vectors containing the desired gene are genetically engineered, so are celled transformed.
How can plants be genetically manipulated?
They can have a gene inserted into their cells which makes them resistant to insect pests.
What is an example of creating insect-resistant plants?
Soybeans are an important food source around the world, yields of soybeans can be greatly reduced due to insect pests that feed on the soybean plant. Scientists have genetically modified soybean plants to include a gene originally found in bacteria Bacillus thuringiensis (Bt). The gene codes for a protein that is toxic to some insects that feed on the soybean :
- Desired gene isolated from Bt with restriction enzymes and inserting it into a plasmid taken from the bacterium Agrobacterium tumefaciens.
- The plasmid is put back into A. tumefaciens.
- The soybean plant cells are directly infected with transformed bacteria. The desired genes get inserted into the soybean plant cells’ DNA, creating a GM plant.
What are positive ethical issues concerning GM soybean plants?
- They reduce the amount of chemical pesticides that farmers use on their crops, which could harm the environment.
- They can be designed to be more nutritious.
What are the negative ethical issues with GM soybean plants?
- Farming GM soybean plants may encourage monoculture (where only one type of crop is planted). Monoculture decreases biodiversity and could leave the whole crop vulnerable to disease, as all the plants are genetically identical.
- A risk that GM soybean plants could interbreed with wild plants creating ‘superweeds’ (weeds that are resistant to herbicides).
What is pharming?
Many pharmaceuticals (medical drugs) are produced using genetically modified organisms, such as animals.
What is an example of producing drugs from animals?
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 protein using goats :
- DNA fragments that code for production of human antithrombin in mammary glands are extracted.
- DNA fragments injected into the goat embryo.
- The embryo is implanted into a female goat.
- The offspring born is tested to see if it can produce antithrombin in their milk.
- 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 (ATryn) that can be given to people with hereditary antithrombin deficiency.
What are 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 people.
What are the negative ethical issues in pharming (in creation of GM animals)?
A concern that manipulating an animal’s genes could cause harmful side-effects to the animal.
Using an animal in this way is enforcing the idea that animals are merely ‘assets’ that can be treated however we choose.
How can scientists use pathogens for research?
Scientists are carrying out research into genetically engineered pathogens (microorganisms that cause disease, such as viruses) in order to find treatments for diseases.
What is an example of how pathogens are used for research?
Scientists found that tumour cells have receptors on their membranes for poliovirus, so poliovirus will recognise and attack them. By genetically engineering the poliovirus to inactivate the genes that cause poliomyelitis (as poliomyelitis is a disease caused by the poliovirus) scientists can use it to attack and kill cancer cells, without causing disease. This may lead to the development of cancer.
What are the positive ethical issues of GM of pathogens to help cure diseases?
It could mean that previously intreated diseases can now be treated, reducing the suffering that they would cause.
What are the negative ethical issues with GM of pathogens to help cure diseases?
- Some people are worried that the scientists that research the pathogens can become infected with live pathogen and cause a mass outbreak of disease.
- Some people are concerned that the GM version of the pathogen could evert back to original form, causing an outbreak of disease.
- Some people worry that in the wrong hands, knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents for biowarfare.
Because of this, researchers using live pathogens have to follow strict protocols, making the chance of any of these happening very, very low.
What is biowarfare?
Deliberately attacking humans or other organisms using biological substances that can harm or cause disease.
What is technology transfer?
The sharing of knowledge, skills and technology
Why may some scientists want a patent for their GM product?
This is because, by law, they can control who uses the product and how for a set period of time.
What are positive ethical issues with patents?
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 can get genetically engineered products faster.
What are the negative ethical issues with patents?
Farmers from poorer countries may not be able to afford patented genetically modified seeds. Even if they could afford it for one year, some patents mean farmers are not legally allowed to plant and grow any of the seeds again without paying again. Many people think this in unfair and that the big companies that own the patents should relax the rules to help farmers in poorer countries.
What are genetic disorders?
Inherited disorders caused by abnormal genes or chromosomes (like cystic fibrosis).
Can gene therapy be used to cure genetic disorders?
It could be used to cure these disorders, but it isn’t used so widely yet. There is a form of somatic gene therapy available, and other treatments are undergoing clinical trials.
What is gene therapy?
Involves altering alleles inside cells to cure genetic disorders.
How do you do gene therapy when the disorder is caused by a mutated dominant allele?
You can ‘silence’ the dominant allele (i.e. by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore.
How do you do gene therapy when the disorder is caused by 2 mutated recessive alleles?
You can add a working dominant allele to make up for them (you ‘supplement’ the faulty gene).
How do you get ‘new’ allele (DNA) inside the cell?
The allele is inserted into cells using vectors. A range of different vectors can be used :
- Altered viruses.
- Plasmids.
- Liposomes (spheres made of lipid).
What are the 2 types of gene therapy?
- Somatic therapy.
- Germ line therapy.
What is somatic therapy?
Involves altering the alleles in body cell, particularly the cells that are most affected by the disorder. It doesn’t affect the individual’s sex cells (sperm or egg) so any offspring could still inherit the disease.
What is an example of somatic therapy?
Cystic fibrosis (CF) is a genetic disorder that’s very damaging to the respiratory system, so somatic therapy for CF targets the epithelial cells lining the lungs.
What is germ line therapy?
Involves altering the alleles in sex cells. This means every cell of any offspring produced from these cells will be affected by gene therapy and they won’t inherit the disease. Germ line therapy in humans is currently illegal.
What are the positive ethical issues of gene therapy?
- Gene therapy could prolong lives of people with life-threatening genetic disorders.
- Gene therapy could give people with genetic disorders a better quality of life if it helps to ease the symptoms.
- Germ line therapy would allow carriers of genetic disorders to conceive (create an embryo by fertilising an egg) a baby without that disorder.
- Germ line therapy could decrease the number of people that suffer from genetic disorders and cancer, which is beneficial for individuals and society as a whole (fewer people require treatment).
What are the negative ethical issues of gene therapy?
- The technology can potentially be used in ways other than for medical treatment, such as treating the cosmetic effects of ageing.
- There is potential to do more harm than good (i.e. risk of overexpression of genes).
- There is a 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 the disadvantages of gene therapy?
- 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 (i.e. cancer).
- An inserted allele could get overexpressed, producing too much of the missing protein, and so causing other problems.
- The effects of the treatment may be short-lived in somatic therapy.
- The patient may have to undergo multiple treatments with somatic therapy.
- It may be difficult to get the allele into specific body parts.
What does gene sequencing mean?
Finding out the order of bases in a gene.
What does genome sequencing mean?
Finding out the order of bases in all of an organism’s DNA.
How can DNA be sequenced using the chain-termination method? What are the 3 steps?
Step 1 : Add a mixture of the following to 4 separate test tubes :
- A single stranded DNA template (the DNA to be sequenced)
- DNA polymerase (the enzyme that joins DNA nucleotides together).
- Lots of DNA primers (short pieces of DNA).
- Free nucleotides (lots of free A,T,C and G nucleotides).
- A fluorescently-labelled modified nucleotide (like a regular nucleotide, but once it’s added to a DNA strand, no more bases are added after it). A different modified nucleotide is added to each tube (A, T, C* and G*).
Step 2 : The tubes undergo PCR which produces many strands of DNA. These strands are different lengths because each one terminates at a different point depending on where the modified nucleotide was added.
Step 3 : The DNA fragments in each tube are separated by electrophoresis and visualised under UV light (because of the fluorescent label). The complementary base sequence can be read from the gel. The smallest nucleotide (1 base) is at the bottom of the gel. Each band after this represents one more base added. So by reading the bands from the bottom of the gel upwards, you can build the DNA sequence one base at a time.
What is genome sequencing?
The chain-termination method can only be used for DNA fragments up to 750 bp long. So if you want to sequence the entire genome (all the DNA) of an organism using this method, you need to chop it up into smaller pieces first. The smaller pieces are sequenced and then put back in order to give the sequence of the whole genome.
What is the method for genome sequencing?
Step 1 : A genome is cut into smaller fragments (about 100,000 bp) using restriction enzymes.
Step 2 : The fragments are inserted into bacterial artificial chromosomes (BACs). These are man-made plasmids. Each fragment inserted into different BAC.
Step 3 : The BACs are inserted into bacteria. Each bacterium contains. BAC with a different DNA fragment.
Step 4 :
What is genome sequencing?
Chain-termination method can only be used for DNA fragments up to about 750 bp long. If you want to sequence an entire genome (all the DNA) of an organism using this method, you need to chop it into smaller pieces first. The smaller pieces are sequenced and then put back in order to give the sequence of the whole genome.
What is the method of genome sequencing?
Step 1 : A genome is cut into smaller fragments (about 100,000 bp) using restriction enzymes.
Step 2 : The fragments are inserted into bacterial artificial chromosomes (BACs). These are man-made plasmids. Each fragment is inserted into a different BAC.
Step 3 : The BACs are inserted into bacteria. Each bacterium contains a BAC with a different DNA fragment.
Step 4 : The bacteria divide, creating colonies of cloned (identical) cells that contain a specific DNA fragment. Together the different colonies make a complete genomic DNA library.
Step 5 : DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA.
Step 6 :
