M6, C21 Manipulating Genomes Flashcards
define PCR (polymerase chain reaction)
Artificial DNA replication. It is a technique which allows DNA fragments of interest to be copied many times (amplification).
This amplified material can then be used for a variety of other genetics techniques.
what reagants are used in PCR
DNA sample to be studied
Primers – short pieces of single stranded DNA. Designed to be complementary to the DNA sequence you are interested in
DNA nucleotides – ACGT bases
DNA polymerase – ‘thermophilic’ version known as Taq. Used because it does not denature at high temperatures
What is the step-by-step process of polymerase chain reaction
1) DNA sample is mixed with, primers, DNA nucleotides and Taq (DNA polymerase).
2) The mixture is heated to 95°C to break the hydrogen bonds holding the complementary strands together, This denatures the double stranded DNA sample to make single stranded DNA.
3) The temperature is reduced to around 55°C in order to allow the primers to bind.
4) This binding forms small sections of double stranded DNA within the DNA sample.
5) The DNA polymerase (Taq) can bind to these double stranded sections.
6) The temperature is raised to 72°C , because this is the temperature at which the DNA polymerase enzyme works best.
7) The DNA polymerase extends the small sections of double stranded DNA by adding free nucleotides to the unwound DNA.
8) When the DNA polymerase reaches the end of the strand, a new double stranded DNA molecule has been generated for only the region you are interested in.
9) The whole process is repeated many times in a cycle, and the DNA replicates.
what is the step-by-step process of gel electrophoresis
1) DNA fragments are treated with restriction enzymes to cut up the fragments
2) DNA samples are placed into wells cut in 1 end of the gel
3) The gel is immersed in a tank of buffer solution and an electric current is passed through the solution for a fixed period of time, usually about 2 hours
4) DNA is negatively charged, so is attracted to the positive electrode
5) DNA fragments diffuse through the gel
6) Shorter lengths of DNA move faster than longer lengths
7) The position of the fragments can be shown by dye stains
8) The fragments can be lifted from the gel for further analysis (Southern Blotting)
9) A nylon sheet is placed over the gel, covered in paper towels, pressed and left over night
10) DNA fragments are transferred to the sheet and can be analysed
what 3 techniques can be used to copy, cut or separate fragments of DNA
Polymerase Chain Reaction (PCR)
Cutting out DNA fragments using restriction enzymes
Gel Electrophoresis
define genome
all of the genetic material of an organism
define satellite DNA
short sequences of DNA that are repeated many times
why does the number of repeats of satellite DNA vary between individuals
different lengths of repeats are inherited from both parents
define DNA profiling
producing an image of the patterns in the DNA of an individual
what are some uses of DNA profiling
- crime scene
- determine the parents of a child
- identifying individuals who are at risk of developing disease
- evolutionary relationships
describe the process of DNA profiling in detail
1) Extracting the DNA - using PCR (refer to other flashcard for the process)
2) Digesting the sample - strands of DNA are cut into small fragments using restriction endonucleases which cut strands in the introns (refer to other flashcard)
- fragments at the end of process include a mixture of mini- and microsatellite regions
3) separating the DNA fragments - using gel electrophoresis (refer to other flashcard) - transferred onto a membrane afterwards by southern blotting
4) Hybridisation - radioactive or fluorescent DNA probes are added in excess to DNA fragments
- bind to complementary strands under particular pH and temp
- DNA probes identify the microsatellite regions (they’re more varied than minisatellite regions)
- excess probes are washed off
5) Seeing the evidence - if radioactive labels were added, x-ray images are taken
- if fluorescent labels were used, membrane is placed under UV light
- fragments give a pattern of bars - DNA profile - unique to every individual (except identical twins)
define genetic engineering
combining DNA from different organisms or different sources
what are organisms called that have had their DNA altered by genetic engineering
what type of DNA do they have
transformed organisms or transgenic
they have recombinant DNA (DNA joined together from different sources)
what are restriction enzymes
they are very specific - they cut at a specific point only
recognises palindromic sequences (the normal sequence and the same sequence which is back-to-front)
they catalyse the hydrolysis reaction breaking the phosphate-sugar backbones of DNA
what two ends can restriction enzymes makes
sticky ends - have a strand of single-stranded DNA which are complementary to each other. They will join with another sticky end but only if it has been cut with the same restriction enzyme
blunt ends - they cut straight across both chains
what is DNA ligase
the enzyme used to catalyse a condensation reaction which joins the phosphate-sugar backbones of DNA together
The 3rd stage of genetic engineering is transferring the vector. How can this happen?
- culture the bacterial cells and plasmids in a calcium rich solution and increase the temp (0 to 40). causes bacterial membrane to become more permeable so the plasmid can enter
- electroporation - small electrical current is applied to the bacteria which makes the membranes very porous so plasmids move into cells
- electrofusion - tiny electric currents are applied to the membranes of 2 different cells. this fuses the cell and nuclear membranes of the 2 different cells together to form a hybrid, containing DNA from both
what are the basic stages of genetic engineering in animals
1) Desired gene obtained by using restriction enzymes or producing DNA from the mRNA
2) Making the recombinant DNA using vectors and ligation
3) Transforming cells - vector carries the gene into the host cell
4) Identifying transformed bacteria using marker genes
step 1 of genetic engineering in animals is obtaining the gene
how can this be done
Cutting out gene from a chromosome using restriction enzymes
or
producing DNA from mRNA of a cell
step 2 of genetic engineering of animals is making recombinant DNA
how is this done
- sticky ends are complementary to each other
- DNA ligase seal sugar-phosphate backbone of gene to plasmid DNA (ligation)
- bacterial plasmids are the vectors - containing a marker gene
- forms recombinant plasmid/DNA
what are vectors (genetic engineering)
-can be plasmids - small circular molecules of DNA
-bacteriophages (virus that infects bacteria)
bacteriophage injects its DNA into the bacterium and the phage DNA (with desired gene) integrates with the bacerial DNA
The last stage of genetic engineering in animals is identifying the transformed bacteria. How is this done?
Marker genes identify bacteria that have taken up the plasmid.
1) marker genes are inserted into the vector at the same time as the gene
2) the marker genes can code for fluorescence which will cause the bacteria that have taken up the plasmid to glow under UV light and be identified
give examples of what you would genetically engineer prokaryotes for
hormones eg. insulin
antibiotics
vaccines
enzymes
Why is it much harder to genetically engineer the DNA of eukaryotic animals than bacteria or plants?
animals cell membranes are less easy to manipulate than plant cell membranes
how do genetically engineer plants
1) cut leaf
2) expose the leaf to bacteria carrying desired gene. also add a marker gene
3) this is carried directly into plant cell DNA
4) expose leaf to an antibiotic to kill cells that lack the new genes
5) wait for surviving cells (gene-altered) to multiply and form a callus (clump)
6) Allow callus to sprout shoots and roots
7) Transfer to soil so fully developed adult plant to grow
Describe how soybeans have been genetically modified to be insect-resistant
A gene found in the bacteria Bacillus thuringienis (Bt) codes for a protein that is toxic to some insects which feed on soybean plants.
The gene is obtained by restriction enzymes.
Give pros and cons for GM crops
PROS
- reduces the amount of chemical pesticides farmers use which would harm the environment and this is cheaper
- increases yield
CONS
- encourages monoculture
- decreases biodiversity - leaves whole crop vulnerable to disease as they are genetically identical
- insects damaged by toxins
what are pharmaceuticals
medicinal drugs
what is pharming
pharmaceuticals that are produced using genetically modified organisms
using animals as models (giving the animals the disease and testing medicines on them)
what are the pros of pharming
- can develop medicines to fight off diseases like cancer
- drugs can be made in large quantities making them more available to people
what are the cons of pharming
- negative ethical issues - shouldn’t be editing genes
- harmful side-effects for the animal
what are the pros of using pathogens in scientific research for genetic engineering
Could mean that previously untreatable diseases can now be treated reducing suffering they cause.
what are the cons of using pathogens in scientific research for genetic engineering
- the scientists researching the pathogens could become infected and cause a mass outbreak of disease
- the genetically modified version of a pathogen could revert back to its original form and cause an outbreak of disease
- in the wrong hands it could be very dangerous and be used to attack humans
what is technology transfer in terms of genetic engineering
scientists around the world share their knowledge, skills and technology to create beneficial genetically modified products at a faster rate
what are the cons of big companies owning genetically engineered organisms
They may have legal protection so if anyone else wants to use it they have to pay. LEDCs may not be able to afford this. Even if they can afford it for a year, some patents mean they’re not legally allowed to grow any seeds from that crop without paying again.
define gene therapy
the treatment of a disease by altering the alleles inside cells
if a genetic disorder is dominant, what gene therapy can be done
Dominant:
You can ‘silence’ the dominant allele by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore
if a genetic disorder is recessive, what gene therapy can be done
Recessive:
You can add a working dominant allele
what are the 2 types of gene therapy
Somatic cell gene therapy
Germline cell gene therapy
what are somatic cells
what does the gene therapy do
A somatic cell is any cell of the body except sperm and egg cells
Involves altering the alleles in body cells
is somatic cell gene therapy permanent or temporary, why?
It is only a temporary solution, the healthy allele will be passed on every time the cell divides, but they have a limited lifespan and are replaced from stem cells, which is where the faulty allele is.
what is happening in somatic cell gene therapy
If the genetic disorder is caused by a faulty allele and codes for a non-functional protein, a functioning copy of the allele can be inserted into the relevant specialised cell.
The functioning protein will be made.
what are germline cells
what gene therapy does it involve
Germline cells - eggs or sperms, or an early embryo immediately after fertilisation
Involves altering alleles in the sex cells
what are some positive ethical issues with gene therapy
- Decrease number of people that suffer from GD
- Carriers of GD might be able to conceive a baby free of disease
- Give a better quality of life for those with GD
- Prolong lives of people with genetic disorders
what are some negative ethical issues with gene therapy
Technology could be used in other ways i.e. cosmetic effects of ageing
Potential to do more harm (over expression of genes)
Expensive - money is better spent of other treatment
what are the disadvantages of somatic cell gene therapy (NOT ETHICAL)
- The effects of the treatment are short lived (somatic cell)
- Patients have to undergo multiple treatment (somatic cell)
- It might be difficult to get the allele into specific body cell
- Body could identify vectors as foreign bodies and start an immune response
- Allele could be inserted into wrong place in DNA, could cause more problems, like cancer
- An inserted allele could get over expressed, producing too much of the missing protein
what are some positives of germline cell therapy
every cell of any offspring produced from these cells will be affected by the gene therapy
If this person reproduced later in life, the allele could be passed on (children not affected by disease)
Better quality of life
define gene sequencing
finding out the order of bases in a gene
define genome sequencing
finding out the order of bases in all of an organism’s DNA
Step 1 of DNA sequencing is mixing what in 4 tubes?
in each tube there is:
- a fluoroscently labelled nucleotide (A in one, C in one, G in one and T in one)
- single stranded DNA template
- DNA primer (short pieces of DNA)
- DNA polymerase
- excess of normal free nucleotides
what is the process of DNA sequencing
1) Each of the 4 tubes undergoes PCR to produces lots of strands of DNA.
2) The primer joins to the template strand at the start and free nucleotides join until at a random point the fluorescent-labelled nucleotide (specific to that tube) joins at a point. At this point the sequencing stops.
For each of the lots of strands in the tube, the fluorescent-labelled nucleotide joins at different points.
3) DNA fragments in each tube are separated by gel electrophoresis and visualised under UV light. You have to read it bottom to top because the smaller fragments travel further down. What you read is the complementary sequence so from that you can determine the original DNA sequence.
what is the process of genome sequencing
1) A genome is cut into smaller fragments using restriction enzymes
2) The fragments are inserted into BACs (bacterial artificial chromosomes). Each fragment is inserted into a different BAC
3) The BACs are then inserted into bacteria
4) The bacteria divide, creating colonies of cloned cells that all contain a specific DNA fragment
5) DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA
6) Each piece of DNA is sequenced, using chain-termination method
7) The pieces are put back in order to give the full sequence from that BAC
8) The DNA fragments from all the BACs are put back in order, by computers, to complete the entire genome.
outline the process of pyrosequencing
1) A section of DNA is cut into fragments, split into single strands and then a strand from each fragment is attached to a small bead.
2) PCR is used to amplify the DNA fragments on each bead.
3) Then each bead is put into a separate well.
4) Free nucleotides added to the wells attach to the DNA strands via complementary base pairing. The 4 different types of nucleotides are added to the wells one after the other for 100 cycles.
5) The wells also contain specific enzymes, which cause light to be emitted when a nucleotide is added to the DNA strand. More than one nucleotide can be added at a time if the bases are the same, so the intensity of the light can vary.
6) Computers analyse the occurrence and intensities of the light emitted in the different wells, after each type of nucleotide is added, and process the info to interpret the DNA sequence.
what does high-throughput sequencing mean
give an example
techniques that can sequence a lot faster than original methods
eg. pyrosequencing
what is synthetic biology
building biological systems from artificially made molecules
eg. energy products and drug products
what is computational biology
what is bioinformatics
using computers to study biology
developing and using computer software that can analyse, organise and store biological data
what does studying the genotype-phenotype relationship mean
predicting an organism’s phenotype by analysing its genotype
amino acid sequencing of an organism’s entire protein complement
what is epidemiology
study of health and disease within a population
considers the distribution of disease and the causes and effects
can compare the genomes of people with the disease to detect particular mutations that could be responsible
how can manipulating genomes be used to understand evolutionary relationships
all organisms evolved from shared common ancestors
closely related species evolved away from each other more recently so share more DNA
whole genomes can be sequenced and analysed using computer software to tell us how closely related different species are
what is DNA barcoding
identifying sections of the genome that are common to all species but vary between them
