Module 6.3 - Manipulating Genomes Flashcards
Define the Polymerase Chain Reaction
A method of artificially amplifying DNA to get many copies of the same sample
Uses of PCR
To make enough DNA to test multiple times (crimes, genetic profiling)
Identifying viral infections (can detect small amounts of viral DNA amongst host DNA - can be used to test for e.g. HIV)
Detecting mutations (DNA analysed to look for mutations that cause genetic disease - can be done in parents/embryos)
Forensic science (small quantities of DNA found at a crime scene can be amplified so that there is enough for DNA profiling)
Tissue typing (donor and recipient tissues can be ‘typed’ to reduce risk of rejection in transplants)
Detection of oncogenes/cancer genes (trying to find specific mutations that caused a cancer can allow more specific medication to be given)
Research (can amplify sources of DNA from fossils for sequencing to study evolutionary relationships - in living species genes which are switched on or off can be studied)
What are DNA primers?
10-20 bases of single stranded DNA
Uses of DNA primers
Sequencing and PCR to bind to sections of DNA so that DNA polymerase can bind as DNA polymerase can’t bind to single strands
PCR key steps and temperatures
Denaturation - 95°C
Annealing - 68°C
Elongation - 72°C
Steps of PCR
Small fragment of DNA to be copied mixed with DNA nucleotides, primers and Taq DNA polymerase (comes from a thermophilic bacteria so will not denature at 95°C, optimum is 72°C)
Heated to 95°C - denaturation
High temperature breaks H-bonds between complementary base pairs in DNA to make 2 single strands of DNA
Temperature cooled to 55°C - annealing
Primers bind to each single strand of DNA at the 3’ end, allowing DNA polymerase to bind to double stranded sections
Heated to 72°C - elongation
Taq DNA polymerase adds DNA nucleotides to single strand according to complementary base pairing rules
This will eventually create a copy of the original fragment of DNA
This process is repeated over and over again and the number of copies of the DNA fragment increases exponentially
Differences between PCR and DNA replication
PCR heat separates complementary strands, DNA replication DNA helicase and girase separate strands
PCR DNA primers needed for polymerase to join and replication to begin, DNA replication DNA primers not needed
PCR does not copy whole chromosome, DNA replication copies whole chromosome
PCR repeats immediately after one cycle done, DNA replication repeats once every cell cycle
PCR Taq DNA polymerase used, DNA replication DNA polymerase used
PCR artificial DNA replication, DNA replication is natural
Similarities between PCR and DNA replication
Copy DNA
Require polymerase
Define electrophoresis
Method of separating and ordering DNA fragments or proteins based on size
Uses of electrophoresis
Fragments can be identified and analysed
Sequencing and DNA profiling
Steps of electrophoresis of DNA
Small amounts of DNA can be amplified using PCR
DNA is cut into smaller fragments using restriction enzymes (the same restriction enzymes must be used to cut the fragments for any of the individuals involved in the identification for forensics)
The fragments are placed into the wells at the end of the gel plate where the negative electrode/cathode will be
The plate is immersed into a tank filled with buffer solution and an electric current is passed through the tank for 1-2 hrs
DNA is negatively charged (due to phosphoryl groups of sugar-phosphate backbone) and so are attracted to the other end of the plate, where the positive electrode/anode is, so the molecules diffuse along the gel to the other end
The shorter fragments move further in the same period of time than the longer ones
The banding pattern is invisible so the DNA must be stained with ethidium bromide and then viewed under UV light to observe the final banding pattern
Steps of electrophoresis of proteins
Done int he same way as DNA
Sodium dodecyl sulphate (SDS) is added to proteins to give them equal negative charge
This means that they can be separated by molecular mass rather than charge
This can be used to analyse proteins by mass in blood to diagnose medical conditions (e.g. sickle cell anaemia, diseases in which patients have higher levels of foetal haemoglobin than they should)
Define DNA profiling
Way of identifying individuals by characteristics of their DNA
Often used to compare DNA of more than one individual
What are Short Tandem Repeats (STRs)?
Loci on the genome composed of 2-10 base pairs which repeat between 5-50 times in a row
The number of repeats at each loci varies from person to person so we can use these to compare the DNA of different individuals
How do we find out the number of STRs a person has at each location?
Electrophoresis
More repeats in STRs = larger DNA fragment = doesn’t move as far in electrophoresis
Steps of creating a DNA profile
DNA obtained for all people to be compared (elf, from saliva/hair)
DNA amplified using PCR
DNA from all people cut into different size fragments using the same restriction enzymes (DNA from different people will be different sizes as the number of repeats in the STR will vary)
DNA fragments separated based on size using electrophoresis, people to be compared are loaded into different wells
Banding pattern examined (small fragments move further) and compared
Uses of DNA profiling
Forensic science (convicting criminals of crimes based on DNA left at crime scenes, identifying body parts in fires/plane crashes)
Maternity/paternity testing (half of child’s DNA, and therefore half the STRs on a DNA profile, is from the mother and half from father)
Studying evolutionary relationships (finding common ancestors between different species, the more similar the banding pattern the more closely related)
Analysis of disease/genetic screening (some diseases are caused by STRs which repeat too many times e.g. Huntington’s)
Advantages of genetic screening
Can identify presence of a disorder Removes uncertainty Allows early treatment May improve life expectancy/quality of life Allows informed choice about having children Allows IVF and embryo screening Allows foetal testing and termination Choice of adoption
Disadvantages of genetic screening
False positives/negatives
Only small number of tests available, not available for all conditions
Presence of gene may not result in condition
Confirmed presence gives stress/fear
Problem telling/testing rest of family
Discrimination by employers/insurers
Ethics of termination
Could increase intolerance/discrimination of disabled
Define DNA sequencing
The process of working out the order of bases on a DNA molecule/gene
Steps of original Sanger method (chain termination method)
Single stranded DNA, DNA polymerase and DNA nucleotides are mixed with one type of radioactively labelled nucleotides (once added to a sequence, terminate DNA synthesis)
DNA sequences of every possible length were created (each terminating with a radioactively labelled nucleotide)
These were run via gel electrophoresis to work out the sequence on the gene
Disadvantages of original Sanger method
Very slow
Labour intensive
Only suitable for very short genes
Steps of updated Sanger method
Fluorescently labelled nucleotides replaced radioactive ones
All 4 types of labelled nucleotides placed together into a sequencing machine (rather than separately)
Machine runs different lengths of DNA through a gel in a capillary tube (instead of gel electrophoresis)
A laser scans each length and reads the fluorescent base sequence as a sequence of colours (each colour specific to each base) to reveal the sequence
Advantages of updated Sanger method
Much faster
Less labour intensive
Steps of pyrosequencing
Nucleotides washed over the DNA in a specific order
When a complementary nucleotide is present it joins the chain
The addition of a nucleotide to the chain releases energy
The energy activates the protein luciferin
Light released by luciferin is detected and recorded on a pyrogram
If two identical nucleotides are added together then the intensity of the light emitted is doubled
Points about high throughput sequencing methods
Much faster at sequencing whole genomes than the Sanger method
Cheaper
More errors tend to be made
Aims of the Human Genome Project
Work out the order/sequence of all the 3 billion base pairs in the human genome
Identify all the genes
Advantages of the Human Genome Project
Improved genetic screening/testing Location of genes that might be linked to increased chances of inheriting a disease New gene therapy New knowledge of how humans have evolved Personalised medicines Synthetic biology
Comparing genes between species
Evolutionary relationships explored by considering similarities in genomes between two species as beneficial genes are conserved by evolution, so this has led to the reclassification of some organisms
Identify genes which have been altered to give rise to differences between organisms (e.g. FOXP2 gene is slightly different in humans so has enabled speech)
Identification of genes common to most/all living things can give clues as to the relative importance of these genes to life
Research differences in gene interaction leading to different proteins being produced
Medical research by comparing genomes of pathogenic and non-pathogenic bacteria to identify genes responsible for causing disease
Comparing genomes between individuals
Epigeneitcs - study of changes in organisms by the modification of gene expression rather than changes of the genetic code
Methylation of DNA can influence gene regulation so mapping the methylation of a whole genome can help our understanding of diseases like cancer developing or not in genetically similar individuals
Investigate the relationship between which genotypes cause which phenotypes
Early human migration can be mapped by comparing genomes of humans from around the world
Medical advances can be made by producing drugs specific to an individual”s genome
How could a genome sequence allow us to predict the structure of a protein?
3 bases = codon
Codon = amino acid
Chain of amino acids = primary structure of protein
This relies on scientists knowing the location of exons (coding region) and introns (non-coding regions)
What is synthetic biology?
Allowed by development of gene sequencing
Designing and building useful biological devices and systems (e.g. producing medication, detecting and cleaning pollution)
Examples of synthetic biology
Biosensors (GE bioluminescent bacteria coat a microchip and glow if petroleum air pollutants are present)
Info storage (scientists can encode huge amounts of digital info onto a single strand of synthetic DNA)
Nanotechnology (produces materials - e.g. amyloid fibres for making bio films for adhesion as microbes will stick to these so they can be sued to clean waste water)
Novel proteins (specially designed - e.g. haemoglobin that wont bind to CO)
Producing medicines (engineering bacteria/fungi to produce the active ingredient of a drug otherwise hard to extract)
Define genetic engineering
Introducing genes from one organism into another
Define recombinant DNA
DNA from 2 different sources joined together
Define transgenic organisms
Organisms which have gene(s) added to their DNA by genetic engineering
4 key stages of genetic engineering
Required gene is obtained
Copy of the gene is placed inside a vector
Vector carries the gene into the recipient cell
Recipient expresses the gene
How to obtain the gene if the base sequence is not known
Isolate mRNA from cells expressing the gene
Use reverse transcriptase to synthesise a single stranded complementary DNA strand (cDNA)
Add DNA primers, DNA polymerase and DNA nucleotides to produce a double strand
Add unpaired nucleotides at the ends to give complementary sticky ends to the ones to be cut on the plasmid
How to obtain the gene if the base sequence is known
Automated polynucleotide synthesiser builds up the gene
PCR primers used to amplify gene
DNA probe used to locate gene which is ut out using restriction enzymes
What is a DNA probe?
A short, single-stranded length of DNA (around 50 bases long) which is complementary to a section of DNA of interest (so it will anneal to it using H-bonds between complementary base pairs)
DNA probes uses
Locate a desired gene for isolation for GE then cut it out using restriction enzymes
Identify the same gene on different genomes for cross species comparison
Identify/screen alleles for genetic diseases
How can DNA probes be labelled?
Radioactive isotopes
Fluorescent markers
What are restriction enzymes?
Restriction endonucleases Cut DNA At restriction sites of specific base pairs (4-6bp long) Leave sticky ends or blunt ends Restriction sites are palindromic
Define vector
Something that carries DNA from one cell to another
e.g. plasmid or weakened virus
How is the gene placed into a vector?
Cut open plasmid using restriction enzymes (same ones used to isolate the gene with)
This will leave complementary sticky ends
Sticky ends of gene and plasmid anneal and H bonds form between bases (A to T and C to G)
Ligase forms phosphodiester bonds between sugar and phosphate groups on backbone to seal it
Recombinant DNA formed
Different ways of getting the vector into the recombinant organism’s cells
Heat shock and calcium salts (bacteria heated and cooled with calcium salts to make membranes porous to take up vector)
Electroporation (high voltage applied to disrupt membrane)
Electrofusion (electrical fields help introduce DNA into cells)
Transfection (DNA packaged into a virus which can then transfect the cell)
Agrobacterium tumefaciens (recombinant Ti/tumour inducing plasmids inserted into the bacterium A. tumefaciens which infects some plants and inserts genome into host genome)
Why would not all bacteria be transgenic?
Ligase may have resealed a plasmid before it became recombinant
Bacteria may not have taken up a plasmid
What is replica plating?
Method of finding which bacteria are now transgenic
What is a genetic marker?
A gene with a known location that can be used to identify individuals (e.g. antibiotic genes used in replica plating)
How to identify which bacteria have taken up recombinant DNA for antibiotic resistance
Plasmids have 2 genes for antibiotic resistance (ab1 and ab2) which act as genetic markers
Restriction enzymes have specific restriction sites in the middle of the ab2 resistance gene
Plasmids mixed with desired gene and ligase
Ligase will reveal some plasmids with the gene and some without
Bacteria take up the plasmids
Bacteria are then replica plated
First grown on standard agar - all colonies grow
Cells from colonies transferred onto agar with antibiotic 1 in it - only cells with plasmids grow
Cells from these colonies transferred onto agar with antibiotic 2 in it - only cells with plasmids that do not have the desired gene will grow
Colonies on plate 1 and not plate 2 have the desired gene and are grown on a mass scale
Explain why a mutated allele may cause a genetic disease
Mutated allele = wrong sequence of DNA bases
Wrong sequence of DNA bases transcribed into wrong mRNA strand
Wrong mRNA strand translated into wrong sequence of amino acids
Wrong sequence of amino acids = wrong polypeptide= wrong shape of protein = wrong/non-functioning protein = symptoms of disease
Define gene therapy
Treating genetic disorders using genetic technology
e.g. diabetes, SCID, Parkinson’s, cystic fibrosis
Somatic cell gene therapy
Gene therapy by inserting functional alleles into somatic/body cells
Introduce new genes by vectors (virus or lipsome) or ex vivo (take cells out, modify then replace)
Augmentation - adding a functional version of a gene so that the correct protein is made (relieves symptoms)
e.g. killing cells - cancerous cells genetically engineered to produce antigens to help immune system recognise cancer cells and destroy them
Disadvantages of using viruses in somatic cell gene therapy
May cause an immune response so person becomes immune to virus and therapy would no longer work
May insert allele into wrong location in the genome, could disrupt other genes and if it disrupts a gene controlling cell division it could increase the risk of cancer
Germline gene therapy
Gene therapy by inserting functional alleles into gametes or zygotes
Germ cells - cells leading to the production of a new organism
All cells in new organism will have desired gene
May pass on desired gene to offspring
Done in animals, illegal in humans
Disadvantages of germline gene therapy
Could introduce a genetic disease/increase risk of cancer by gene being inserted into wrong location
Permanent changes to human DNA that can be passed on (considered unethical)
Could lead to eugenics (‘breeding out’ disease, disabilities or ‘undesirable characteristics’)
Patient cannot give consent to DNA modification
Differences between somatic cell and germline gene therapy
Harder to deliver genes in somatic cell therapy (has to be ex vivo or in vectors which can be ineffective), straight into germ cells for germline gene therapy
Somatic cell therapy can’t pass on new genes to offspring
Somatic cell therapy specialised cells are treated and don’t divide so can’t pass on genes to other cells so need to repeat gene therapy regularly as specialised cells are replaced, germline has no need to repeat therapy as every cell and hence every new cell will contain a copy of new genes
Somatic allowed in humans, germline not allowed in humans for ethical reasons
Suitable vector for transgenic mammals
Virus
Liposome
Suitable vector for somatic gene therapy
Virus
Liposome
Suitable vector for transgenic plants
Agrobacterium tumefaciens
Ti plasmid
Suitable vector for transgenic bacteria
Plasmid
Why is it easier to perform gene therapy when the normal allele is the dominant allele of the gene concerned?
Has an effect when added to the genome
Not masked
No need to remove/inactivate the recessive/mutant allele
Advantages of genetic manipulation of animals
Pharming - getting GM animals to make pharmaceuticals (e.g. some proteins are too big for bacterial cells to make so GM animals are used to make them in their milk)
Alpha antitrypsin can be made by GM goats to treat hereditary emphysema
Disadvantages of genetic manipulation of animals
Modification is not done with animal welfare in mind so animal could be uncomfortable/harmed
Advantages of genetic manipulation of plants
Insect resistance in GM soya - Bt gene inserted into soya plant DNA, they produce the Bt toxin, killing insects that eat them and would reduce crop yield
No need to use pesticide - prevents possibly contaminating wild plants with insecticide/prevents human exposure when spraying insecticide
Herbicide resistance in GM soya - gene resistant to herbicide inserted into DNA, crop can be sprayed, all weeds killed but crop not damaged, can reduce number of sprays needed
Disadvantages of genetic manipulation of plants
Could crossbreed with wild plants and pass on insect/herbicide resistance, could reduce biodiversity, wild plants may kill insects in the food chain, super weeds resistant to herbicide could be made
GM seeds are often patented do farmers may not legally be able to clone/fertilise/collect seeds of the plants they grow but would have to buy new seeds each year, often poor farmers need GM crops the most (e.g. golden rice, drought resistant plants), raises ethical questions about availability of this technology
Advantages of genetic manipulation of microorganisms
GM pathogens grown in labs for research into diseases, their metabolism and drugs to treat them
Viruses are modified to be harmless to be vectors in somatic cell gene therapy
Disadvantages of genetic manipulation in microorganisms
GM pathogens could escape labs and cause epidemics
