Manipulating Genomes (OCR A Level Biology): Flashcards

A term describing multiple DNA sequencing technologies that allow the simultaneous sequencing of multiple DNA strands, making them rapid and able to produce large datasets very quickly.

• Each type of dideoxynucleotide is labelled with a specific fluorescent dye

• The single-stranded chains are separated by mass using electrophoresis inside a capillary tube

• A laser beam illuminates the dideoxynucleotides and a detector reads the colour and position of each fluorescence

• The detector feeds the data into a computer for storage or analysis

The newest high-throughput sequencing methods, e.g. nanopore sequencing and pyrosequencing.

• Evolutionary biology and classification

• Personalised medicine

• Disease diagnosis

Its length — shorter fragments travel further, so a single-nucleotide fragment travels furthest and each band above represents one more base added.

They do not use electrophoresis, and most are automated rather than manually interpreted.

False — each tube contains only one type of dideoxynucleotide (A, C, T or G).

True

All of the genes within an organism.

A field of biology that involves the storage, retrieval, and analysis of data from biological studies (such as data on DNA, RNA and protein sequences, and the relationship between genotype and phenotype).

• The genetic code is used to predict the amino acid sequence within the protein

• From the amino acid sequence, scientists can predict how the protein will fold into its tertiary structure

They are needed to create and store the large databases that hold information about organisms' gene sequences and amino acid/protein sequences.

Bioinformatics is used to compare an organism's genome with the genomes of others in databases. A greater degree of similarity between genomes indicates that the organisms are more closely related.

An organism whose genome and biology can be studied to gain insight into humans or other species.

The genomes of many individuals of the same species are sequenced and compared. A species with high genetic variation will show a large number of differences in base sequences between individuals.

The genomes of different species are compared. Species with a small number of differences between their genomes are likely to share a more recent common ancestor than species with a large number of differences.

The protein cytochrome c is involved in respiration and is found in a wide range of species, making it especially useful for comparisons between different species.

Specific genes are "knocked out" (their expression is stopped) and the effect on the organism's phenotype is observed. Knowing the genome sequence allows scientists to target specific base sequences to knock out.

• Highly infectious strains can be identified (e.g. the Delta variant of SARS-CoV-2)

• A pathogen's ability to infect multiple species can be investigated (e.g. Ebola infecting primates as well as humans)

• The most appropriate control measures can be implemented

• Potential antigens for use in vaccine production can be identified

Any single organism may have anomalies/mutations in its DNA sequence that are atypical of the species. Using DNA from multiple individuals produces a reference genome that is representative of the species.

Public funding meant there were no commercial interests or influence over the project.

Correlations have been found between changes in specific genes and the likelihood of developing certain inherited diseases. For example, mutations in the BRCA1 and BRCA2 genes substantially increase the risk of breast cancer, and specific genes have been linked to Alzheimer's disease.

The nematode worm Caenorhabditis elegans, used to study organ development, neurone development and cell death. It was the first multicellular organism to have its genome fully sequenced, and is useful because it has fewer than 1000 cells and is transparent.

Making the data publicly available meant it could be shared rapidly between researchers and used by anyone, maximising the benefit to humans.

True

False — it is created from the DNA of multiple individuals, so it is representative of the species and not skewed by one individual's anomalies.

The complete set of genes (the entire DNA sequence) present within a cell or organism.

The full range of proteins that a cell or organism is able to produce.

Sections of DNA within the genome that do not code for proteins.

Large amounts of non-coding DNA are present in the genome, and it is very hard to distinguish these non-coding sections from the coding DNA.

• The presence of regulatory genes\n\n- The process of alternative splicing

Regulatory genes control gene expression, and therefore influence which proteins are synthesised.

It allows a single gene to be spliced in different ways, producing multiple different proteins from that one gene.

Two main reasons:\n\n- Alternative splicing, which allows a single gene to code for more than one protein\n\n- Post-translational modification of proteins, which produces different functional proteins after translation

In the Golgi apparatus.

The proteome is larger than the genome, due to alternative splicing and post-translational modification of proteins.

Alternative splicing allows a single gene to produce multiple different proteins.

True — alternative splicing and post-translational modification let one gene give rise to more than one protein.

False — it often takes place in the Golgi apparatus.

A recent area of research that aims to create new biological parts, devices and systems, or to redesign systems that already exist in nature.

The genetic code can be used to predict the amino acid sequence within a protein.\n\nOnce the amino acid sequence is known, scientists can predict how the new protein will fold into its tertiary structure.

It involves large alterations to an organism's genome, rather than small changes.\n\nThis new genome can cause a cell to operate in a novel way, not yet seen before in nature.

• Using existing DNA sequences\n- Using entirely new sequences (designed and written with special computer programmes so that they produce specific proteins)

The most well-known use of synthetic biology is the commercial production of the antimalarial drug artemisinin.

Artemisinin was first isolated in China from the native plant Artemisia annua.

A. annua is difficult to cultivate.\n\nThis led to an unstable supply of artemisinin at an ever-changing price, often too expensive for those needing the drug most.

• They constructed a DNA sequence for a whole new metabolic pathway containing genes from bacteria, yeast and *A. annua\n- This pathway produces artemisinic acid, a precursor to artemisinin\n- The pathway can be inserted into yeast cells, which then produce artemisinic acid\n- The precursor is then converted into artemisinin using an inexpensive process*

A precursor to artemisinin produced by the engineered metabolic pathway inserted into yeast cells; it is later converted into artemisinin using an inexpensive process.

It allows scientists to design new DNA sequences that produce specific proteins, so that engineered cells can carry out desired functions such as producing useful drugs.

False — it can also use entirely new sequences, designed and written with special computer programmes.

True

An in vitro method of DNA amplification used to produce large quantities of specific fragments of DNA (or RNA) from very small starting quantities.

It can produce billions of identical copies of the DNA or RNA sample within a few hours, which can then be used for analysis.

• DNA profiling (e.g. identifying criminals or determining paternity)

• Genetic engineering

• Target DNA (or RNA) to be amplified

• Primers (forward and reverse)

• DNA polymerase (usually Taq polymerase)

• Free nucleotides

• Buffer solution

Short sequences of single-stranded DNA with base sequences complementary to the 3' end of the DNA being copied. They define the region to be amplified by telling DNA polymerase where to begin building the new strands.

Taq polymerase comes from the thermophilic bacterium Thermus aquaticus, so:

• It does not denature at the high temperature (95°C) used in the denaturation stage.

• Its optimum temperature is high, which prevents annealing of DNA strands that have not yet been copied.

Free nucleotides are used to construct the new DNA (or RNA) strands.

A thermal cycler, which automatically provides the optimal temperature for each stage and controls the length of time spent at each stage.

1. Denaturation

1. Annealing

1. Elongation / Extension

The double-stranded DNA is heated to 95°C, which breaks the hydrogen bonds holding the two DNA strands together, separating them into single strands.

The temperature is decreased to between 50–60°C so that the forward and reverse primers can anneal to the ends of the single strands of DNA.

The temperature is increased to 72°C (for at least a minute), the optimum temperature for Taq polymerase to build the complementary strands, producing new identical double-stranded DNA molecules.

In each PCR cycle the amount of DNA is doubled, so a standard run of 20 cycles produces around a million DNA molecules.

The buffer solution provides the optimum pH for the reactions to occur.

True — it comes from the thermophilic bacterium Thermus aquaticus, so it stays active at 95°C.

False — annealing occurs at 50–60°C; 95°C is used during the denaturation stage.

A technique used widely in the analysis of DNA, RNA and proteins, in which molecules are separated according to their size/mass and their net charge.

Molecules are separated according to:\n\n- Their size/mass\n\n- Their net charge

Positively charged molecules move towards the cathode (negative pole).\n\nNegatively charged molecules move towards the anode (positive pole).

DNA is negatively charged due to its phosphate groups.

The gel contains tiny pores, so:\n\n- Smaller molecules move quickly (further from the wells)\n\n- Larger molecules move slowly (remain closer to the wells)

The gel typically used for separating DNA fragments is agarose, whereas polyacrylamide (PAG) is used for proteins.

Different gels have different sized pores, which affect the speed at which the molecules can move through them.

1. Create an agarose gel plate in a tank, with wells cut into the gel at one end\n\n1. Submerge the gel in an electrolyte solution (a salt solution that conducts electricity)\n\n1. Load the DNA fragments into the wells using a micropipette\n\n1. Apply an electrical current, with the negative electrode connected to the end holding the wells so DNA moves towards the anode\n\n1. Smaller/shorter fragments move faster and further than larger fragments\n\n1. Transfer the fragments onto absorbent paper/nitrocellulose, add probes, then use an X-ray or UV light to produce a pattern of bands

Regions found in the non-coding part of DNA that contain variable numbers of repeated DNA sequences and vary between different people (except identical twins). Also called 'satellite' or 'microsatellite' DNA.

Restriction endonucleases (enzymes) are used to cut the DNA into fragments.\n\nDifferent restriction enzymes cut at different base sequences, so scientists use enzymes that cut close to the VNTR regions.

Single-stranded DNA sequences that are complementary to the VNTR regions being sought. They carry a means of identification: either a radioactive label (which darkens X-ray film) or a fluorescent dye (which fluoresces under UV light).

Proteins are prepared by:\n\n- Denaturing them (to break the disulfide bonds)\n\n- Then manipulating them into rod shapes (which are negatively charged) to allow separation by size

The charge of the R groups of amino acids depends on the pH.\n\nBuffer solutions keep the pH constant, ensuring the proteins carry a consistent charge during separation.

DNA moves towards the anode (the positive pole), because it is negatively charged.

True

False — DNA is negatively charged, so it moves towards the anode (positive pole).

A technique that produces an image of an individual's variable number tandem repeats (VNTRs) as a pattern of bars, used to identify individuals — for example to identify suspects of a crime or to identify corpses. Every person (except identical twins) has a unique profile.

Short, repeating regions of non-coding DNA (around 20 to 50 bases long). The number of VNTR regions is inherited from your biological parents, and the length of the VNTR regions is unique to each individual (except identical twins).

Because the length of the VNTR regions is unique to each individual.

The number of VNTR regions is inherited from your biological parents.\n\nThis is why the more closely related two people are, the more likely their repeat patterns are to be similar.

• Obtain the DNA (e.g. from the root of a hair, a spot of blood, semen or saliva)\n\n- Amplify the DNA using PCR to produce large quantities from a very small sample\n\n- Cut the amplified DNA into fragments using restriction endonucleases (which cut close to specific VNTR sequences)\n\n- Separate the fragments by length using gel electrophoresis\n\n- Add radioactive or fluorescent probes that are complementary to, and bind to, specific VNTR regions\n\n- Produce X-ray images (radioactive probes) or use UV light (fluorescent probes) to reveal the pattern of bars\n\n- Analyse the resulting pattern of bars (the DNA profile)

To amplify the DNA — it produces large quantities of the required DNA fragment from very small samples (even from just one molecule of DNA or RNA).

They cut the amplified DNA into fragments. Different restriction endonucleases cut close to different VNTR sequences.

It separates the DNA fragments according to their length/size, so that the VNTRs (which are unique to each person except identical twins) can be compared.

Radioactive or fluorescent probes are added; these are complementary to, and bind to, specific VNTR regions.

Look for the profile with the most bands in common with the crime scene sample.

• Samples of body cells or fluids (e.g. blood, saliva, hair, semen) are taken from the crime scene or victim\n\n- The DNA is extracted and profiled\n\n- The profile is compared with samples from the suspect (or a criminal DNA database), the victim, and control samples from people unconnected to the crime\n\n- Care must be taken to avoid contamination of samples\n\n- It can also identify bodies or body parts that are otherwise unidentifiable (e.g. badly decomposed remains)

• Identifying individuals at risk of developing particular diseases (certain VNTR sequences are associated with diseases such as cancers and heart disease)\n\n- Determining familial relationships, e.g. paternity cases or immigration cases\n\n- Species conservation — supporting captive breeding programmes to reduce the chance of inbreeding

Look for the combination of the parents' bands that together cover all of the child's bands.

True

False — DNA profiling analyses non-coding regions (the VNTRs).

The manipulation of the DNA sequences of an organism.

The genetic code is universal.

This means almost every organism uses the same four nitrogenous bases – A, T, C and G (with a few exceptions).

The same codons code for the same amino acids in all living things.

This means that genetic information is transferable between species.

Altered DNA that has had nucleotides from a different source introduced into it (typically a different species).

An organism that contains nucleotide sequences from a different species.

Any organism that has had genetic material introduced into it.

By combining lengths of nucleotides from different sources (typically from different species).

The altered DNA produced is called recombinant DNA (rDNA).

The mechanisms of transcription and translation are also universal.

This means the transferred DNA can be transcribed and translated within the cells of the GMO.

If an organism contains nucleotide sequences from a different species it is called a transgenic organism.

A form of genetic engineering that involves the transfer of fragments of DNA from one organism/species into another.

The resulting organism contains recombinant DNA and is a genetically modified organism (GMO).

A field of science that studies the design and construction of new biological pathways, organisms and devices, as well as the redesigning of existing natural biological systems.

• Genetic modification of crops to increase yield through resistance to drought, disease, pesticides and herbicides, or to increase nutritional value (e.g. golden rice)

• Genetic modification of livestock to give disease and pest resistance and increased productivity

• Genetic modification of bacteria to produce medicines (e.g. insulin), decompose toxic pollutants or carry out large-scale chemical production

True

False — a transgenic organism contains nucleotide sequences from a different species.

• Identification of the DNA fragment or gene

• Isolation of the desired DNA fragment (using restriction enzymes, a gene machine or reverse transcriptase)

• Multiplication of the DNA fragment (using the polymerase chain reaction, PCR)

• Transfer into the organism using a vector

• Identification of the cells that have taken up the new DNA fragment (using a marker), which are then cloned

An enzyme used to cut genes (DNA) at specific base sequences known as restriction sites. Different restriction enzymes cut at different restriction sites.

An enzyme used to join together the cut ends of DNA by forming phosphodiester bonds.

An enzyme used to build double-stranded DNA from a single-stranded RNA template.

A carrier used to deliver DNA fragments into a cell. Examples include plasmids, viruses and liposomes.

• 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

They are the three methods used to obtain (isolate) the desired gene:

• Restriction enzymes cut the gene out of DNA at specific restriction sites

• A gene machine synthesises the gene artificially

• Reverse transcriptase builds double-stranded DNA from an mRNA template

It is used to encourage the uptake of plasmid vectors into cells, by making the cell membrane more permeable to the plasmid DNA.

A gene that codes for an identifiable substance that can be tracked, used to identify cells that have successfully taken up the new DNA fragment.

• Fluorescent markers e.g. green fluorescent protein (GFP), which fluoresces under UV light

• Enzyme markers e.g. β-glucuronidase (GUS), which transforms colourless or non-fluorescent substrates into coloured or fluorescent products

• Antibiotic resistance markers - the required gene is inserted into an antibiotic resistance gene

The required gene sequence is inserted into a gene for antibiotic resistance, which inactivates that resistance gene.

This means successfully transformed bacteria will be killed if exposed to the antibiotic.

A replica plating method is therefore used to identify and isolate the successfully transformed bacteria.

Ligase joins together the cut ends of DNA by forming phosphodiester bonds.

The DNA fragment is multiplied using the polymerase chain reaction (PCR) before being transferred into the organism.

Liposomes fuse with cell membranes to transfer DNA into cells.

False — restriction endonucleases cut DNA at specific restriction sites; ligase joins the cut ends by forming phosphodiester bonds.

True

A legal right that gives the owner the ability to prevent others from replicating their invention (e.g. a genetic modification) for a limited period of time.

Microorganisms such as bacteria, yeast, or animal cells.

They are used for research and for treatments (e.g. diabetes, cancer, infectious diseases, haemophilia).

Eukaryotic cells carry out the post-translational modification required to produce a suitable human protein.

This is because they possess the Golgi apparatus and/or the necessary enzymes, which prokaryotic cells lack.

• More cost-effective to produce large volumes (unlimited availability)

• Simpler and faster to produce many proteins

• A reliable supply is available

• Proteins can be engineered to be identical to human proteins or given beneficial modifications

• Avoids moral, ethical or religious concerns about using cow- or pork-derived proteins

In 1982, insulin was the first recombinant human protein to be approved for use, in the treatment of diabetes.

• Restriction endonucleases cut open bacterial plasmids and DNA ligase splices the human insulin gene into the plasmid

• The recombinant plasmids are inserted into Escherichia coli by transformation (calcium ion bath, then heat or electric shock)

• Transgenic bacteria are identified using markers, then isolated, purified and placed in fermenters with optimal conditions

• The bacteria multiply by binary fission and express insulin, which is extracted and purified

• Identical to human insulin, unless deliberately modified (e.g. to act faster or more slowly)

• A reliable supply to meet demand, without depending on meat stock

• Fewer ethical, moral or religious concerns (not extracted from cows or pigs)

• Fewer rejection problems, side effects or allergic reactions

• Cheaper to produce in large volumes

• Useful for people with animal insulin tolerance

• Organisms with the desired characteristics are produced more quickly

• All organisms will contain the desired characteristic (no chance of a recessive allele arising in the population)

• The desired characteristic can come from a different species/kingdom

• Resistant to herbicides – increases productivity / yield

• Resistant to pests – increases productivity / yield

• Enriched in vitamins – increases nutritional value (e.g. rice modified to produce β-carotene for vitamin A)

The Bt toxin gene, taken from the bacterium Bacillus thuringiensis, causes the soya plant to produce its own insecticide.

When an insect ingests the plant, the alkaline conditions in its gut activate the toxin, killing the insect. The toxin is harmless to vertebrates because their stomachs are highly acidic.

The genetic modification of livestock (e.g. "biopharm" sheep and goats) to produce useful human proteins, typically secreted in their milk — for example the human blood protein AAT in sheep's milk, or antithrombin in goat's milk.

A growth hormone gene from a chinook salmon was combined with a promoter gene from an ocean pout (a cold-water fish).

The promoter keeps the growth hormone continually expressed throughout the year (not just in spring/summer), so the salmon reaches market size in half the time, increasing yield.

They are not cell-specific or species-specific, so they can infect the cells of many mammals.

Specific genes are removed so the virus cannot replicate inside host cells, which also creates space for the insertion of desired genes.

• GM seeds are more expensive and do not "breed true", so farmers must rebuy seeds yearly — a struggle in developing countries

• Lack of long-term research on effects on human health (e.g. allergies, toxicity); without labelling, consumer choice is removed

• Pollen may contaminate nearby non-GM/organic crops

• Reduced biodiversity and risk of GM crops becoming weeds or "superweeds" via herbicide-resistance gene transfer

• Antibiotic-resistance marker genes could transfer to pathogens, creating a "superbug"

• Tampering with viral genomes could create a novel or more harmful virus

They are grown in culture.

All the salmon are female and sterile.

False — eukaryotic cells are preferred because they carry out the post-translational modification needed for a functional human protein.

True

The use of various mechanisms to alter a person's genetic material in order to treat, or cure, diseases.

• Replace a faulty gene

• Inactivate a faulty gene

• Insert a new gene

Scientists are having difficulty:

• Finding delivery systems that can transfer normal alleles into a person's cells

• Ensuring the gene is correctly expressed once it is inside the cells

The delivery system used to transfer genetic material (a normal allele) into a person's cells.

They already have the mechanisms needed to recognise cells and deliver genetic material into them.

Examples of non-viral vectors being researched for gene therapy include liposomes and 'naked' DNA.

Changes in genetic material are targeted to specific body cells and do not target the gametes (germ cells), so the changes cannot be passed on.

The effects on somatic cells are also often short-lived.

Ex vivo – the new gene is inserted via a virus vector into the cell outside the body. Blood or bone marrow cells are extracted, exposed to the virus, grown in the laboratory and returned to the person by injection into a vein.

In vivo – the new gene is inserted via a vector into cells inside the body.

Any changes made to the genetic material of germ cells are potentially permanent and could therefore be inherited by future generations.

The body's inability to produce adenosine deaminase (ADA), an enzyme key to the functioning of the immune system.

Without this enzyme, children can die from common infections and often need to be kept isolated in plastic 'bubbles'.

A virus transfers a normal allele for ADA into T-lymphocytes removed from the patient, and the cells are then returned to the patient via an injection.

The treated T-lymphocytes are replaced by the body over time, so the patient requires regular transfusions (every three to five months) to keep their immune system functioning.

Retroviruses insert their genes randomly into the host's genome. This means the gene could be inserted into another gene or a regulatory sequence, which could result in cancer.

Initial treatments caused cases of leukaemia in children, so researchers switched to lentiviruses or adeno-associated viruses.

Doctors inject adeno-associated viruses containing the normal alleles of one of the genes that damages the photoreceptors directly into the retina.

Patients who have had the injections have shown an improvement in their eyesight.

False — somatic gene therapy targets body cells, not the gametes, so the changes cannot be inherited.

False — adeno-associated viruses do not insert their genes into the host genome, so the genes are not passed on to daughter cells during cell division.