Mr G bio 6 Genome projects and Gene technologies Flashcards
what is a genome?
Complete set of genes in an organism.
what must happen before gene sequencing methods can be used?
Gene sequencing methods only works on fragments of DNA, so to sequence the entire genome of an organism, the DNA must be chopped up first.
Smaller pieces are sequenced and then put back in order to give the sequence of the whole genome.
what is the proteome?
range of proteins an organism can produce
why is sequencing proteomes easier in simple organisms?
Bacteria do not have non-coding DNA.
Easy to determine their proteome from the DNA sequence of their genome.
why is sequencing proteomes in simple organisms useful in medical/scientific research?
identifies protein antigens on the surface of disease causing bacteria and viruses – vaccines.
Also allows scientists to monitor pathogens during an outbreak of a disease – leads to better management of the spread of infection and can help identify antibiotic resistance factors.
why is translating a genome into a proteome difficult in complex organisms?
-contain large sections of non-coding DNA
-contain compex regulatory genes, which determine when the genes that code for particlar proteins are turned on and off
makes it hard to find parts of genome that code for proteins
what were sequencing methods like in the past?
labour- intensive, expensive and could only be done small scale
how have new sequencing techniques developed?
now techniques are automated, more cost effective and can be done on a large scale. scientists can now sequence whole genomes much more quickly
what does recombinant DNA technology involve?
Recombinant DNA technology involves transferring a fragment of DNA from one organism to another.
what is recombinant DNA technology used for?
Because the genetic code is universal, and because transcription and translation mechanisms are universal, the transferred DNA can be used to produce proteins in the cells of the recipient organisms.
Donor and recipient organisms do not have to be the same species.
what is a transgenic organism?
organisms that contain transferred DNA
what is needed before you can transfer a gene from one organism to another?
need to get a DNA fragment containing the gene that you are interested in (target gene)
what are the 3 methods of making DNA fragments?
-Using Reverse Transcriptase
-Using Restriction Endonuclease Enzymes
-Using a Gene Machine
why is it difficult to obtain a DNA fragment containing the target gene?
as most cells only contain 2 copies of each gene. BUT cells that produce the protein coded for by the target gene will contain many mRNA molecules which are complementary to the gene – so mRNA is easier to obtain.
how can you use reverse transcriptase to make a DNA fragements?
-Reverse transcriptase, makes DNA from an RNA template.
-The DNA produced is called complementary DNA (cDNA).
-mRNA must be isolated first, then mixed with free DNA nucleotides and reverse transcriptase, making cDNA.
how are restriction endonuclease enzymes able to make DNA fragments?
-Some sections of DNA have palindromic sequences of nucleotides.
-These sequences consist of antiparallel base pairs (base pairs that read the same in the opposite direction).
when can restriction endonucleases be used?
-If recognition sites are present at either side of the DNA fragment you want, you can use restriction endonucleases to cut the fragment out from the rest of the DNA.
-The DNA sample is incubated with the specific restriction endonucleases, which cuts the DNA fragment out via a hydrolysis reaction.
what happens after the DNA fragment is cut out using restriction endonucleases?
Sometimes the cut leaves sticky ends – small tails of unpaired bases at each end of the fragment.
Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has complementary sticky ends.
what does a gene machine allow?
fragments of DNA can be synthesized from scratch, without the need for a pre-existing DNA template – made from the knowledge of the primary sequence
Instead, a database contains all the necessary information to produce the DNA fragment.
This means that the DNA sequence does not have to exist naturally – any sequence can be made.
what is the process of using a gene machine?
1.The sequence is designed.
2.The first nucleotide in the sequence is fixed to a support e.g. a bead.
3.Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching.
4.Short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. Once these are complete, they are broken off from the support and all the protecting groups are removed. The oligonucleotides can then be joined together to make longer DNA fragments.
what are the two different ways of making many copies of a target gene?
In vivo cloning – gene copies are made within a living organism. As the living organism grows and divides, it replicates the DNA making many copies of the gene.
In vitro – gene copies are made outside a living organism using polymerase chain reaction (PCR).
what is a vector?
something that’s used to transfer DNA into a cell – plasmids or bacteriophages.
what is the process of using In vivo cloning to make recombinant DNA?
1.Vector DNA isolated.
2.Vector DNA is cut open using the same restriction endonucleases that was used to isolate the target gene.
Causes sticky ends of the vector and the target gene to be complementary.
3.Vector DNA and DNA fragment are mixed with DNA ligase.
Ligase joins the sticky ends of the DNA fragment and vector DNA – ligation, by complementary base pairing.
4. Vector DNA + DNA fragment = Recombinant DNA
after recombinant DNA is made during in vivo cloning, what is part 2 (transforming cells)?
- recombinant DNA transfers the target gene into the host cells
- host cells that take up the vector containing the target gene are said to be transformed
3.if a plasmid is used, host cells have to be persuaded to take in recombinant plasmid
what do bacteriophages do?
infect the host bacteria and inject its DNA, which is then integrated into the bacterial DNA
what is part 3 of in vivo cloning? (Idenitfying transformed cells)
1.Marker genes are inserted into the vectors at the same time as the target gene. All transformed host cells will therefore contain the marker and the target gene.
2.Host cells are grown on agar plates and each cell divides and replicates its DNA, producing a colony of cloned cells.
3. Identified transformed cells are allowed to grow more, producing many copies of the cloned gene.
for transformed host cells to produce the protein coded for by the target gene, what must the vector contain?
specific promoter and terminator regions
what is a promoter region?
DNA sequences that tell RNA polymerase where to start producing mRNA
what is a terminator region?
tell RNA polymerase where to stop producing mRNA
what is the process of in vitro cloning?
1.Reaction mixture – DNA sample, free nucleotides, primers and DNA polymerase.
2. DNA mixture is heated to 95oC to break hydrogen bonds between the two stands of DNA. So the DNA strands separate.
Mixture is then cooled to between 50-65oC so that the primers can bind (anneal) to the strands.
3. Reaction mixture is heated back up to 72oC, so DNA polymerase can work. DNA polymerase joins DNA nucleotides together to make DNA. Specific base pairing means new complementary strands are formed.
4. Two new copies of the fragment of DNA are formed and once cycle of PCR is complete.
The cycle is repeated by heating the mixture to 95oC and this time all 4 strands are used as templates
how can a transformed microorganism be produced?
made using the same tech as in vivo cloning
how can transformed plants be produced?
1.A gene coding for a desirable protein is inserted into a plasmid.
2.Plasmid is added to a bacterium which is then used as a vector to get the gene into the plant cells.
If the promoter region has been added along with the gene, the transformed cells will produce the desired protein
how can transformed animals be produced?
-A gene coding for a desirable protein is inserted into an early animal embryo or into an egg.
-If inserted into a very early embryo, all the body cells of the resulting transformed animal will end up containing the gene.
-Inserting the gene into the egg means that when a female reproduces, all the cells of her offspring will contain the gene.
what are the advantages to using gene tech in agriculture?
-Transformed crops may give higher yields/ more nutritious – reduces famine and malnutrition
-Transformed crops may be resistant to pests so fewer pesticides are used, reducing costs and any environmental problems associated with using chemicals
-Transformed crops may be resistant to drought so can be grown in areas with little rainfall.
what are the concerns about using gene tech in agriculture?
-Monocultures – whole crop is vulnerable to the same disease because they are all genetically identical.
-Monocultures – reduce biodiversity.
-Superweeds – weeds that are resistant to herbicides. Made by transformed crops interbreeding with wild plants.
what are the benefits to using gene tech in terms of industry?
Enzymes can be produced using transformed organisms which are then often used in industrial processes – large quantities for less money.
what are the concerns with using gene tech in terms of industry?
-Labelling – will consumers have the choice to choose between products made using genetically modified organisms.
-processes used to purify proteins from GMOs, could lead to the introduction of toxins into food industry.
-A few large biotechnology companies control some forms of genetic engineering. As use of this tech increases, these companies get bigger and more powerful, forcing out smaller businesses – Anti-globalization companies are against this.
benefits of using gene tech in medicine?
1.Drugs and vaccines can be produced using recombinant DNA technology.
2.Insulin
3.Drugs made using recombinant DNA tech produced cheaper, quickly and in large quantities – more affordable and available to more people.
what is the purpose of gene therapy?
Gene therapy involves altering the defective genes (mutated alleles) inside cells to treat genetic disorders such as cancer.
how can gene therapy be used to reverse two mutated recessive alleles?
you can add a working dominant allele to make up for them – you ‘supplement’ the faulty ones.
how can gene therapy be used to reverse a mutated dominant allele?
‘silence’ the dominant alleles by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore.
what are the two types of gene therapy?
somatic and germ line
what is somatic gene therapy?
altering the alleles in body cells, particularly the cells that are most affected by the disorder.
Somatic therapy doesn’t affect the individual’s sex cells – offspring can still inherit the disease.
what is germ line therapy?
altering the alleles in the sex cells.
Every cell of the offspring produced from these cells will be affected by the gene therapy and they won’t suffer the disease.
what are the ethical issues surrounding gene therapy?
Technology could be used for non-medical treatments such as cosmetics of ageing.
Might do more harm than good – risk of overexpression of genes – gene produces too much of the missing proteins.
what are DNA probes used for?
DNA probes can be used to locate specific alleles of genes (on a chromosome)
Helps to identify if a person’s DNA contains a mutated allele that causes a genetic disorder.
how are DNA probes visible?
-using autoradiography
-using UV light
what is the process of using DNA probes?
1.Sample of DNA digested into fragments using restriction enzymes. And separated using electrophoresis.
2.Separated DNA fragments are transferred to a nylon membrane and incubated with a fluorescently labelled DNA probe. If the allele is present, the DNA probe will bind (hybridise) to it.
3.Membrane is exposed to UV light and if the allele is present there will be a fluorescent band.
how can mutliple alleles be screened at once using DNA probes?
1.A sample of fluorescently labelled human DNA is washed over the array.
2.If the labelled human DNA contains any of the DNA sequences that are complementary to any of the probes, it will bind to the array.
3.This means you can screen the DNA for different mutated genes at the same time.
4.The array is washed to remove any fluorescently labelled DNA that hasn’t stuck to it, and then visualized under UV light.
5.Any labelled DNA attached to a probe will show up.
6.Any spot that fluoresces means that the person’s DNA contains that specific allele.
what are the uses of screening with DNA probes?
Help identify inherited conditions.
Determine whether a patient will respond to specific drugs and create personalised medicine
Identify health risks
Genetic screening may lead to discrimination by insurance companies and employers
genetic counselling- advising patients about the risks of genetic disorders
what are VNTRs?
Not all of an organism’s genome codes for polypeptides.
Some of the genome consists of variable number tandem repeats (VNTRs) – base sequences that repeat over and over but do not code for proteins.
The number of times these sequences repeat differs from person to person, so the length of these sequences in nucleotides differs too.
