✨Module 6: Manipulating genomes Flashcards
What are the 2 types of DNA sequencing?
Sanger sequencing
High throughput sequencing
DNA sequencing method/chain termination/Sanger sequencing technique!
1). Single stranded DNA for sequencing is mixed with DNA polymerase, DNA primers, excess free nucleotides ACTG, and terminator bases, each with a coloured fluorescent tag (ACGT). Test tubes are placed in a thermocycler and PCR begins.
2). Primer anneals to the ends of the single stranded template.
3). At optimum temp, DNA polymerase brings complementary bases to the single stranded DNA template.
4). At any time, DNA polymerase can insert one of the terminator bases by chance which results in the termination of DNA replication as no more bases can be added.
5). As the terminator bases are present in low amounts and are added at random places, this results in many DNA fragments with varying length. After many cycles, all possible DNA chains will be produced.
6). DNA fragments are separated according to length by gel electrophoresis. Lasers detect the fluorescent tags on the terminator bases and learn the order of bases in complementary DNA. From this the original strand can be deciphered.
Because each of the test tubes only contains one type of terminator base, it is possible to know what the terminal nucleotide of each fragment is.
Explain why terminator bases are so important in the Sanger method and in the modern high-throughput sequencing methods.
=> All possible length DNA fragments are synthesised.
=> Having different coloured fluorescent tags attached to 4 different terminator bases allow us to work out the original sequence of DNA.
=> High throughput sequencing is much more complex and rapid, but relies on terminator bases to terminate chains in final stages.
What is high-throughput sequencing? (don’t need to know the details).
Multiple DNA sequencing technologies that allow simultaneous sequencing of multiple DNA strands.
High throughput methods are rapid and so produce large datasets very quickly.
Why is it faster to sequence genomes now than in the olden days?
Originally, each stage was carried out by hand in the lab. But modern techniques involved machines and many DNA fragments could be processed at once.
Discuss how DNA sequencing has changed the ways in which we identify species and our understanding of evolutionary relationships.
Before, species identification was done by observation of anatomical and physiological features. With DNA sequencing, genome similarities are examined and comparisons made to standard species genome, which is much more accurate.
Evolutionary relationships - DNA sequencing looks at the difference in mutations between species. By calculating average mutation rate, you can calculate when two species diverged.
Define genome.
Contains all genes in an organism.
What is the human genome project?
An international and collaborative research program that collects DNA samples from many individuals of a species and are used to create a reference genome. More than 1 individual is used, as one individual may have mutations in DNA sequence.
How can sequencing DNA determine protein sequences?
The genetic code can be used to predict the amino acid sequence within a protein. Then they can predict how the polypeptide will fold into its tertiary structure.
Describe the function of spliceosomes.
Enzymes present in eukaryotic nuclei that remove the introns from the mRNA and fuse the exons together before translation into polypeptide.
The spliceosomes may join the same exons in a variety of ways, which would code for different amino acids, different proteins, different phenotypes.
Describe bioinformatics.
Involves storage and analysis of data (in large databases) such as DNA/RNA sequences, genotype-phenotype relationship. It allows scientists to analyse these large amounts of data generated during sequencing of billions of base pairs.
Bioinformatics allows for comparisons with genomes of other organisms. Can identify similarity between organisms to see how closely related they are => evolutionary relationships.
Data is displayed in ways that make sense and help identify patterns.
How can bioinformatics be used to investigate:
1. Genetic variation
2. Evolutionary relationships
3. Genotype-phenotype relationship
4. Epidemiology
- Many individuals of same species have their genomes sequenced and compared. A large difference in one organism means they have high level of genetic variation.
- Individuals of different species have their genome sequenced. Species with a small difference share more recent common ancestor.
- Involves ‘knocking out’ different genes/stopping their expression and observing the effect it has on the phenotype of an organism.
- Genomes of pathogens can be sequenced and analysed to aid research and disease control. Can produce antigens for vaccines.
Describe computational biology.
Uses the data from bioinformatics/different databases to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances. Working out the 3D structure of proteins, sequencing billions of base pairs.
Explain how analysing the genomes of pathogens helps.
=> Doctors can identify antibiotic resistant bacteria, ensuring antibiotics are only used when they’ll be effective and preventing spread of antibiotic resistance.
=> Doctors can track the progress of an outbreak - each strain of a pathogen has a slightly different genome and so can be accurately identified by DNA sequencing, which means place of origin/individuals with disease can be identified.
What is synthetic biology? Describe the different techniques of synthetic biology.
A recent area of research that aims to:
=> Create new biological parts and systems that already exist in nature.
=> It goes beyond genetic engineering, as it involves large alterations to an organisms genome.
=> Synthesis of new genes to replace faulty genes e.g. in cystic fibrosis.
Polymerase chain reaction/PCR purpose.
A small piece of DNA is amplified many times to produce large amounts of DNA or RNA. It is used for DNA profiling/criminal investigations and genetic engineering. It is known as in vitro (happens outside the organism) method of DNA amplification.
Describe some uses of DNA profiling and the benefits.
=> Identify criminals to prove innocence or guilt. PCR is performed on traces of DNA left at crime scenes obtained from skin/hair. This DNA profile is compared to a sample taken from the suspect.
=> Determining paternity.
=> DNA profiling also identifies individuals who are at risk of developing particular disease. Certain non-coding microsatellites have been associated with increased risk.
=>Tiny amounts of DNA can be used.
Discuss the limitations of DNA profiling.
=> Could ignore other evidence in criminal cases and mistakes could be made.
=> Contamination of samples with DNA from other organisms.
What is an intron?
Large non-coding region of DNA that is removed from mRNA before it is translated into a polypeptide chain.
Explain how PCR works.
- Reaction mixture is set up with DNA sample, free nucleotides, primers and DNA polymerase.
- DNA mixture is heated to 95 degrees C to break the hydrogen bonds. DNA polymerase doesn’t denature at this temp - many cycles of PCR can be carried out without having to use new enzymes each time.
- Mixture is cooled to 50-60 degrees so primers can anneal (bind) to the ends of the strands.
- Reaction mixture is heated to 72 degrees C, so DNA polymerase can work in optimum temp. The DNA polymerase lines up free DNA nucleotides alongside each template strand. Complimentary base pairing means new strands are formed.
- Two new copies of the fragment of DNA are formed and one cycle of PCR is complete, then the cycle starts again. Mixture heated to 95 degrees C and this time all 4 strands are used as template strands.
Each PCR cycle …
Doubles the amount of DNA. 1,2,4,8 strands.
Explain how you can use restriction enzymes to get DNA fragment by PCR.
Some sections of DNA have palindromic sequences of nucleotides. Have antiparallel base pairs (base pairs read in opposite directions).
What are primers in PCR?
Short sequences of single stranded DNA that have a complementary base sequence to the DNA sample being copied. They bind to the ends. They define the region that is to be amplified by telling DNA polymerase where to begin building new strands.
What is special about the DNA polymerase in PCR?
The enzyme used to build the new DNA/RNA is called Taq polymerase as it comes from thermophilic bacterium. This means it won’t denature at high temps involved in first stage of PCR. Also so its optimum temp is high enough to prevent annealing of DNA strands that haven’t been copied yet.
Why is a buffer used in PCR?
They provide optimum pH for enzymes.
What is the purpose of gel electrophoresis?
To separate DNA fragments based on size/length using an electric field. It can be used to show genotypes of individuals by separating polypeptide chains produced by different alleles.
What happens during gel electrophoresis?
Molecules are separated based on mass/size and overall charge.
=> Positively charged molecules move towards cathode (-)
=> Negatively charged molecules moves towards anode (+)
DNA is negatively charged due to phosphate groups and move towards anode.
Small molecules/short fragments move quickly and further from the wells, larger molecules move slowly.
Different gels have different sized pores which affect the speed at which molecules can move through them.
We use gel electrophoresis after PCR. How do we prepare the fragments for electrophoresis?
After amplification, restriction endonucleases are used to cut the DNA into fragments. Different restriction endonucleases cut DNA at specific nucleotide sequences, known as restriction sites. All restriction endonucleases make 2 cuts: each through the double helix. Scientists will use specific endonucleases to cut the DNA close to the variable number tandem repeat regions.
Tandem repeats are regions found in non-coding DNA/introns. These DNA sequences vary between different people (except identical twins).
METHOD: Gel electrophoresis.
- Insert DNA fragments into wells at the end of the agarose gel in a tank using a micropipette. Before make sure the agarose gel plate is submerged into an electrolyte solution (salt solution that conducts electricity).
- Apply electrical current to tank. Cathode must be connected to the end of the plate with the wells as the DNA fragments will move towards the anode.
- The gel is then immersed in alkali in order to separate the DNA into single strands. This is then transferred on a nylon membrane by Southern blotting. This nylon membrane is covered with several sheets of dry absorbent paper. the single stranded DNA is transferred onto the membrane by capillary action.
- Hybridisation - radioactive or fluorescent DNA probes are added to label the fragments. These are short DNA or RNA sequences that are complementary to the DNA sequence. They bind under particular conditions of pH and temp. DNA probes identify microsatellites.
- Membrane with radioactively labelled DNA fragments is placed onto an X-ray film. This reveals dark bands where the radioactive DNA probes have attached.
If fluorescent labels were added to the DNA probes, the membrane is placed under UV light so the fluorescent tags glow.
What are probes and why do we add them after gel electrophoresis?
Single stranded DNA sequences that are complementary to the tandem repeats. These can either be radioactive labels or fluorescent dyes.
Why can gel electrophoresis be used to separate proteins?
Proteins are polymers of amino acids, which are different sizes and are charged molecules. They would move through gel during electrophoresis with an electric current in the same way as nucleotides do – and they would similarly be separated by size and charge.
What is genetic engineering?
Practical technique of isolating genes for desirable characteristics in one organism, and placing them into another organism using a vector.
What is a transgenic organism?
An organism that contains a gene from another organism. Aka genetically modified organism (GMO).
Explain the process of genetic engineering using restriction endonucleases.
- Restriction endonucleases cut the required gene from DNA. Many endonucleases cut the 2 DNA strands unevenly, creating sticky ends. These sticky ends make it easier to insert the desired gene into the DNA of another organism.
- The same restriction endonuclease is used to cut the plasmid so the plasmid has complementary sticky ends to the ones on the DNA fragment. Once these line up, DNA ligase forms phosphodiester bonds between sugar and phosphate groups on 2 DNA strands, joining together.
After these, bacteria multiply in a fermenter and produce insulin. Insulin can be extracted and treat people with diabetes.
*Multiplication of the DNA fragment (using polymerase chain reaction - PCR)
*Identification of the cells with the new DNA fragment (by using a marker), which is then cloned.
Explain the process of genetic engineering with extracted mRNA.
- Extracting mRNA that codes for insulin. mRNA is treated with the enzyme reverse transcriptase to make complementary DNA (cDNA). This technique makes it easier to identify the desired gene.
- Plasmid is obtained from bacteria, plasmid cut with restriction enzyme, plasmid and cDNA fuse by DNA ligase. Plasmid is the vector.
How is the vector/plasmid transferred to the host cell/bacterium?
The plasmid with recombinant DNA is transferred to host cell by transformation. Two methods:
=> Culture bacterial cells and plasmids in a calcium-rich solution and increase temp. This causes the bacterial cell membrane to become permeable and plasmids can enter.
=> Electroporation where a small electric current is applied to bacteria to make the membrane permeable so plasmids can enter.
What 3 enzymes are required for genetic engineering?
Restriction endonucleases - used to cut genes at SPECIFIC base sequences (restriction sites). This creates sticky ends.
DNA ligase - used to join together the sticky ends of DNA by forming phosphodiester bonds.
Reverse transcriptase - used to build double stranded DNA from single stranded RNA.
Describe how scientists ensure that they an identify bacteria that have the plasmid and gene.
Plasmids have gene for antibiotic resistance, so bacteria that take up engineered plasmids can be identified.
Plasmid also contains marker gene - usually fluorescence or enzyme which changes colour of medium.
What is the purpose of using a selectable marker in a plasmid during genetic engineering?
To identify cells that have successfully taken up the recombinant DNA.
What is the purpose of using a promoter sequence in genetic engineering?
To initiate transcription of inserted DNA
Describe another method of genetically modifying an organism.
Electrofusion where tiny electric currents are applied to membranes of 2 different cells so the cells fuse together to form a hybrid or polyploid cell, containing DNA from both. This method is used to produce GM plants.
Electrofusion is different in animal cells, which don’t fuse easily as plant cells. But electrofusion is important in making monoclonal antibodies.
Genetically engineering prokaryotes.
Bacteria have been genetically modified to produce insulin, antibiotics, vaccines.
Genetically engineering plants.
A desired gene e.g. for pest-resistance, higher yield is placed in plasmid along with the marker gene like antibiotic resistance. This is then carried directly into the plant cell DNA. The transgenic plant forms a callus which is a mass of GM plant cells, each of which can be grown into a new transgenic plant.
Transgenic plant cells can also be produced by electrofusion.
Genetically engineered animals.
Animal cell membranes are much harder to manipulate than plant cells.
Describe some uses of genetic engineering.
Genetic modification of:
=> Crops to increase crop yield through resistance to drought, disease, pesticides.
=> Livestock to give disease and pest resistance.
=> Bacteria to produce medicines e.g. insulin. Additionally bacteria can be modified to decompose toxic pollutants.
What are the types of vectors you can use in genetic engineering?
They are used to deliver DNA fragments into a cell:
Plasmids - transfer DNA into bacteria or yeast
Viruses - transfer DNA into human cells or bacteria
Liposomes - fuse with cell membranes to transfer DNA into cells
What 2 types of marker gene could be used in the plasmid in genetic engineering?
Fluorescent markers e.g. green fluorescent protein (GFP) which fluoresces under UV light.
Antibiotic resistance marker genes - The required gene sequence is inserted into a gene for antibiotic resistance. This inactivates the antibiotic resistance gene and therefore means that successfully transformed bacteria will be wiped out if exposed to the antibiotic. A replica plating method is then used to isolate the successfully transformed bacteria
Advantages of using recombinant insulin?
=> Identical to human insulin unless modified to have different properties, so fewer rejections
=> Reliable supply to meet demand (no need to depend on the availability of meat stock)
=> Fewer ethical, moral or religious concerns (proteins are not extracted from cows or pigs)
Discuss the impact of GM crops such as soya beans.
Scientists have inserted a gene into soya beans so they produce Bt protein. It’s toxic to many pests that attack the plant.
GM crops could reduce the impact of farming on the environment due to there being less need to spray pesticides.
GM crops will help feed the population.
However, whenever antibiotic resistance is used as a marker gene to create GM crops, there is a risk that this resistance could spread to wild populations of plants and into bacteria.
Describe some pros and cons of GM crops.
Crop plants have been genetically modified to be:
Herbicide resistant – reduces competing weeds and increases yield. Biodiversity could be reduced if overused.
Pest resistant – reduces amount of pesticide spray, protecting the environment and helping poor farmer. Increases yield of crops. BUT non-pest insects may be damaged by toxins in GM plants. Insect pests may become resistant to pesticides in GM crops.
Disease resistant - increases yield, transferred genes might spread to wild populations.
Example of a genetically modified animal?
Swine fever-resistant pigs - scientists inserted a gene from wild African to European pigs, giving them immunity.
Describe some ethical issues around genetic engineering.
=> GE animals used to produce pharmaceuticals (pharming).
=> GE pathogens can be produced for research.
=> GE seeds would be hard to acquire for poor farmers (patenting and technology transfer).
Explain how pharming is an ethical problem.
This involves the production of human medicines.
=> Creating animal models by adding or removing genes so animals develop certain diseases.
=> Altering animal DNA so they produce proteins used in human medicine.
Why is there little debate about the ethics of genetically engineered MICROORGANISMS?
They’ve been used safely for many years.
Produce many beneficial materials like insulin.
What is gene therapy? What are the 2 types?
Replacing/inactivating a faulty allele (one that codes for a genetic disease) with a normal allele.
Somatic and germ line cell gene therapy.
Somatic cell gene therapy.
Replacing a mutant allele with a healthy one in affected somatic (body cells)/target cells.
Cons - short term and needs repeating. healthy allele will be passed on every time the cell divides by mitosis but somatic cells have a limited life. Eventually they are replaced by stem cells which will have the faulty allele and will be passed onto children.
Somatic doesn’t affect the gametes.
Germ line cell gene therapy.
Inserting a healthy allele into germ cells/embryonic cells. Healthy allele would be passed onto offspring. Permanent.
This therapy is successful with animal embryo’s but is illegal for human embryos => potential impact on individuals due to interfering with germ cells. No consent of baby. May enable people to choose desirable/cosmetic characteristics of their offspring.
What are short tandem repeats?
Regions of non-coding DNA that contain repeats of the same nucleotide sequence. They contain variable regions known as polymorphisms, therefore are unique to each individual, making them useful in DNA profiling.
