21 - Manipulating Genomes Flashcards
What type of people have identical DNA?
Identical twins
What makes up an organism’s genome?
All the genetic material it contains (i.e. in eukaryotes mitochondrial DNA would be included)
What is an exon?
Region of DNA which is expressed and codes for a protein
What is an intron?
Sections of DNA which don’t code for amino acids. Introns are removed during protein synthesis.
What is satellite DNA?
Short DNA sequences which are repeated many times
What is minisatellite DNA?
A region where 20-50 base pairs is repeated 50 to several hundred times
What is another name for minisatellite regions?
Variable Number Tandem Repeats (VNTR)
In what 3 parts of DNA are satellite regions found?
Introns, centromeres, telomeres
What is microsatellite DNA?
A region of 2-4 bases repeated 5-15 times
Is satellite DNA coding or non-coding?
Non-coding
What is another name for microsatellite regions?
Short Tandem Repeats (STR)
Satellite regions always appear in the same position on what?
Chromosomes
What is DNA profiling?
Producing an image of the patterns in the DNA of an individual
What are the 5 main stages of DNA profiling?
- Extracting the DNA 2. Digesting the sample 3. Seperating the DNA fragments 4. Hybridisation 5. Seeing the evidence
Roughly what percentage of human DNA is introns?
98%
What are telomeres?
Regions of DNA at the end of chromosomes which do not code for making proteins, and which shorten each time replication occurs
What are centromeres?
The part of a chromosome that links sister chromatids during mitosis
Why does the number of STRs an individual has vary?
Lengths are inherited
What regions of DNA are examined during DNA profiling?
Satellite DNA
What technique is used to create a larger DNA sample for profiling?
PCR (Polymerase Chain Reaction)
What occurs during the extraction stage of DNA profiling?
DNA is extracted from a tissue sample
What are 2 samples which could be used to provide DNA for profiling?
- Cheek swab 2. Blood sample
What does the cutting in DNA profiling?
Special enzymes called restriction endonucleases
What is the site where a restriction endonuclease cuts a sample called?
Restriction site
How many cuts does each restriction endonuclease make?
2- one through each strand of the DNA helix
Why are a mixture of different restriction endonucleases used during DNA profiling?
To produce a mixture of different intact satellite regions
What happens in the second stage of DNA profiling?
Restriction endonucleases are used to cut up the sample
What technique is used to separate DNA fragments in DNA profiling?
Electrophoresis
What technique is used to transport the DNA from the electrophoresis gel to the nylon membrane?
Southern Blotting
What is a DNA probe?
A short, single-stranded section of DNA that has a label attached to make it easily identifiable, and which attaches to a complementary DNA sequence under particular pH or temperature conditions
What do DNA probes identify?
Microsatellite regions
What happens in the hybridisation stage of DNA profiling?
DNA probes attach to complementary DNA fragments, and excess probes are then washed off
What 2 types of label are generally added to DNA probes?
Radioactive and fluorescent tags
Are radioactive or fluorescent DNA tags more commonly used today?
Fluorescent
What does the final result of DNA profiling look like?
A pattern of bars which represents an individual’s DNA profile
How are radioactive DNA probes identified?
The membrane is x-rayed
How are fluorescent DNA probes identified?
The membrane is placed under a UV light so that they glow
What are the 3 steps of PCR?
- Separating the strands 2. Annealing primers 3. DNA synthesis
How does electrophoresis work?
The gel box has an electric field with a positive and negative end. DNA is negatively charged so it migrates to the positive end.
How is fragment size identified using electrophoresis?
It is easier for smaller molecules to move through the mesh of the box, so these travel further
What happens in the 1st stage of PCR?
Temperature increased to 90-95⁰C for 30 seconds to denature DNA
What happens in the 2nd stage of PCR?
Temperature decreased to 50-60⁰C and primers anneal to ends of DNA
What do primers do in PCR?
Allows DNA polymerase to add bases to them to build up complementary DNA strands identical to original sequence
What enzyme is used in PCR?
Taq polymerase
What happens in the 3rd stage of PCR?
Temperature increased to 72⁰C for at least 1 minute to allow taq polymerase to add bases to primers
What are 3 uses of DNA profiling?
- Crime scene investigation 2. Paternity testing 3. Identify individuals at risk of genetic diseases
What was the original method of DNA sequencing?
Sanger sequencing
How is the DNA for sequencing generated in Sanger sequencing?
By entering DNA into a bacterial plasmid
What was one refinement of the Sanger sequencing process?
Swapping radioactive labels for fluorescent ones
What are 4 disadvantages of Sanger sequencing?
- Very expensive 2. Slow 3. Can only be done 900 base pairs at a time 4. Essentially just ‘shotgunning’
What are 4 advantages of Next Gen sequencing?
- Much cheaper 2. Much quicker 3. Can sequence whole genome at once 4. Multiple genomes can be processed at once
What 2 things sped up the human genome project?
- Development of newer, more powerful computers 2. Automation of sequencing techniques
What is a terminator base?
A modified base which stops DNA synthesis
What are terminator bases given?
Fluorescent tags
What 4 things is DNA for sequencing mixed with in the 1st step of DNA sequencing?
- Terminator bases 2. Excess of normal bases 3. Primer 4. DNA polymerase
What are the 4 ingredients placed into in the second stage of DNA sequencing?
A thermal cycler
What happens at 96 degrees celcius in DNA sequencing?
The DNA strands separate into single strands
What happens at 50 degrees celcius in DNA sequencing?
Primers anneal to end of DNA strands
What happens at 60 degrees celcius in DNA sequencing?
DNA polymerase starts to build up new strands
What is the importance of terminator bases in DNA sequencing?
As they are incorporated at random, many different fragments of DNA of different lengths are created, so after many cycles every possible DNA fragment with the reaction terminated at every base will have been created
How are DNA fragments separated in DNA sequencing?
By length using capillary sequencing (basically gel electrophoresis in capillary tubes)
What are the fluorescent markers on terminator bases used for?
To identify the final base of each fragment
What does the order of terminator bases in capillary tubes show?
The order of the complimentary strand of DNA, which can in turn be used to identify the sequence of the template strand
Where are fragments sequenced in next-gen sequencing?
On a plastic slide
What two things about genome sequencing have fallen over the years?
- Time to sequence 2. Cost to sequence
What is bioinformatics?
The development of software and computational tools needed to organise and analyse raw biological data
What are 3 examples of things developed in bioinformatics?
- Statistical tests 2. Mathematical modelling 3. Algorithms
What is computational biology?
Using computers and the processed data provided by bioinformatics to build theoretical models of biological systems
What are 2 things computational biology gives us a better understanding of?
- 3D structures of molecules such as proteins 2. DNA sequences
What is genomics?
Using DNA sequencing methods and computational biology to study genomes
What is the Genomes Project UK10K?
Project to try to get 10,000 genomes from the UK sequenced
What 2 things can genomics reveal patterns in?
- Vulnerability to diseases 2. Inherited DNA
Why is genomics not the miracle identifier for what causes most genetic diseases?
Most are polygenic anyway
What are 4 things sequencing pathogen genomes enables?
- Doctors to find source of infections 2. Doctors to identify antibiotic resistant strains of bacteria 3. Scientists to track progression of diseases and monitor potential epidemics 4. Scientists to discover regions of pathogen DNA for drug targeting or markers for vaccines
What has DNA sequencing allowed in the field of phylogeny?
Evolutionary trees to be built with previously impossible accuracy
How has DNA sequencing aided phylogeny?
Basic mutation rate of different species’ DNA can be calculated, so we can see how long ago 2 species diverged from a common ancestor
What is the challenge for scientists wanting to use DNA sequencing to aid species identification?
To get stock sequences for each species
What is DNA barcoding?
Identifying regions of DNA common to all species, but which vary between them- enabling comparisons to be made
What happens in the International Barcode of Life (IBOL) project?
Scientists identify species using relatively short sections of DNA from a conserved region of the genome
What is the common section of DNA used for animals in the IBOL project?
A 648-base pair region of mitochondrial DNA in gene for cytochrome c oxidase
What is important about the section of DNA used to identify animals in the IBOL project?
It is small enough to be sequenced quickly and cheaply, but varies enough to give clear differences between species
Why is the region of DNA used for animals not also used for land plants in the IBOL project?
It does not evolve quickly enough to show differences between plant species
What is the common section of DNA used for plants in the IBOL project?
Two regions of DNA in chloroplasts
What charge does DNA have and why?
Negative, because of the phosphate group
What is proteomics?
The study and amino acid sequencing of an organism’s entire protein complement
What evidence is emerging about the link between genomes and proteins?
The amino acid sequence is not necessarily what would be predicted from the genome sequence alone due to some genes coding for multiple proteins
What is removed from pre-mRNA?
Introns and, sometimes, some of the exons as well
What are spliceosomes and what do they do?
Enzyme complexes which join exons which are to be translated
How can spliceosomes affect what proteins are coded for?
They can join exons together in multiple different ways, so the same pre-mRNA could produce multiple different proteins
How can proteins be modified after translation?
Cutting and combining of polypeptide chains and addition of phosphate or carbohydrate molecules by other proteins
What is synthetic biology?
Design and construction of new and biological parts; redesign and novel use of biological things for useful purposes
What are 4 techniques of synthetic biology?
- Synthesis of an entire new organism 2. Synthesis of new genes to replace faulty ones 3. Use of whole or partial biological systems in industrial contexts (i.e. immobilised enzymes) 4. Genetic engineering
What is genetic engineering?
The direct manipulation of genes for practical purposes.
Better understanding of what 2 fields has enabled successful genetic engineering?
Genomics and proteomics
What is an organism which carries a gene from another organism called?
Transgenic or a GMO
What do the basic principles of genetic engineering involve?
Isolating a gene for a desirable characteristic from one organism and placing it into another using a specific vector
What is the first stage of genetic modification?
Isolating the desired gene
What is the main technique used to isolate a gene for genetic modification?
By using restriction endonucleases to cut out the desired gene
Why is the same restriction enzyme used to cut both the gene from the target and a gap in the vector?
To create complimentary ends in the plasmid and desired gene
What is a sticky end?
A sequence of unpaired bases on a double-stranded DNA molecule that readily base pairs with a complementary strand. In genetic modification, can be found on the plasmid and inserted gene
What is a blunt end?
Where the restriction endonuclease leaves no bit of single-stranded DNA, so the end piece of DNA is double stranded
What is another technique to isolate a gene for genetic modification apart from using restriction endonucleases?
Getting a bit of mRNA for the desired gene and using reverse transcriptase to make the desired gene
What is an advantage of using the reverse transcriptase method of isolating a gene for genetic modification?
Makes it easier to identify desired gene
What is the DNA produced using reverse transcriptase called?
cDNA (complementary DNA)
What are the most commonly used vectors in genetic engineering?
Bacterial plasmids
What is used to fuse a plasmid and cDNA?
DNA Ligase
What is recombinant DNA?
DNA produced by combining DNA from different sources- for example, combining a bacterial plasmid with cDNA
What is transformation?
Moving the gene vector into the bacterium during genetic engineering
What is electroporation?
A method to introducing recombinant DNA into cells. Brief electrical pulse is applied, creating temporary holes in the plasma membrane and making it more permeable to vectors.
What is a method for checking for plasmid uptake in genetic engineering?
Adding a green fluorescent protein marker gene to the plasmid before uptake so you can use UV light to check uptake
What was a previous method of checking plasmid uptake before GFP and why isn’t it used any more?
Giving the plasmid an antibiotic resistance gene so any bacteria which don’t take it up can be killed off. Obviously no longer used due to concerns about antibiotic resistance.
What is electrofusion?
Using an electrical current to merge two cells and their respective genomes
What is a cell known as when it has multiple, merged genomes?
Polyploid cell
What types of cell does and doesn’t electrofusion work on currently?
Works well on prokaryotes and plant cells, not so much on animal cells
What is electrofusion important in the production of?
Monoclonal antibodies
Is it easier to carry out genetic engineering in prokaryotes or eukaryotes?
Currently prokaryotes
What are 6 examples of useful substances currently produced by genetically modified bacteria?
- Insulin 2. Clotting factors for haemophilia 3. Human growth hormone 4. Antibiotics 5. Pure vaccines 6. Enzymes used in industry
What are 2 methods of genetically modifying plants?
- Electrofusion 2. Using the bacterium Agrobacterium tumefaciens
How can plants be genetically modified using electrofusion? (4 steps)
- Removal of cell wall using cellulases 2. Electrofusion to form polyploid cell 3. Use of plant growth hormones to create new cell wall 4. Callus formation
What are the 5 steps of genetically modifying plants using A. tumefaciens?
- Cut leaf 2. Expose leaf to bacteria containing desired gene and antibiotic resistance gene, and allow bacteria to deliver genes into leaf cells 3. Use antibiotic to kill off remaining bacteria and cells without new genes. Wait for callus to form callus 4. Allow callus to sprout roots and shoots 5. Transfer new plant to soil
What are 3 advantages of crop GM?
- Quicker growth 2, More nutrients in crops 3. Disease and pesticide resistance can be achieved
What are DNA libraries?
Where fragments of DNA created during DNA sequencing are stored in the genomes of microorganisms
What can DNA libraries serve as sources of DNA fragments for?
Further genetic engineering applications or further study of their function
GM microorganisms are used in research of what 3 things?
- Novel medical treatments 2. Industrial processes 3. Development of gene technology
Why are GM pathogens not generally used for research?
Health and safety of researchers and the public, concern it could be used for biological warfare
Where are most GM crops found right now?
The USA, there is only 1 in Europe
What are 4 examples of crops which have already been genetically modified?
Rice, cotton, soy, corn
What are 4 things crops have been genetically modified to be or do?
- Resist drought and extreme heat 2. Contain Vitamin A (golden rice) 3. Produce insecticidal toxin 4. Resist herbicides
Is there any contemporary debate about the use of GM microorganisms?
Not really apart from the modification of pathogens for use in warfare
What are 2 wide-scale hopes for GM plants?
- Feed expanding human population 2. Overcome environmental issues
What is an economic concern about the use of GM crops?
People can be mugged off by corporations using patent laws, especially those in less developed countries who theoretically benefit most from genetic modification
What are 2 vectors for animal tissue currently under research?
- Modified viruses 2. Microinjections (tiny particles of DNA covered in gold)
What are 2 examples of GM animals?
- Swine-fever resistant pigs 2. Faster growing salmon
What is pharming?
Genetically modifying animals to produce medicines and other useful substances for humans
What are the two aspects of pharming?
Creating animal models (i.e. giving animals diseases genetically so they act as models for human treatment), creating human proteins
What is somatic cell gene therapy?
Gene therapy by inserting functional alleles into body cells to replace faulty ones (in a born person)
What are 4 positives of human gene therapy?
- Can prolong lives of those with genetic disorders 2. Can give genetic disease sufferers better quality of life 3. If germ line, sufferers can have baby without fear it gets condition or cancer 4. If germ line, could decrease number of people with disorder
What are 9 negatives of human gene therapy?
- Effects of somatic may be short-lived 2. May need multiple treatments in somatic 3. Might be difficult to get alleles into specific body cells 4. Inserted allele could be overexpressed and lead to overproduction of protein in question 5. Could cause immune response to vector 6. If allele inserted in wrong place could cause problems such as cancer 7. Technology might not just be used for medicine i.e. could be for cosmetic treatment 8. Could do more harm than good 9. May be too expensive and may rob funding from other, more proven technologies
What are 3 drawbacks of somatic cell therapy?
- Sufferers can still pass it on to their kids 2. Only temporary as somatic cells have limited life, and are eventually replaced by stem cells with healthy allele 3. Difficult to get vector into cell and get gene expressed
What is germ line cell gene therapy?
Inserting a healthy allele into a germ cell (usually an ovum) or an embryo immediately after fertilisation as part of IVF
What are 3 ethical objections to germ line cell gene therapy?
- Done to unborn individual, and thus without consent 2. Effects not yet fully known or studied 3. May eventually allow people to choose desirable or cosmetic characteristics in their kids
