8B - Genome Projects and Gene Technologies Flashcards
What is a genome?
An entire set of an organism’s DNA, including all of the genes.
How does sequencing of an entire genome work and why?
- DNA is cut up into small sections, which are then sequenced and assembled again
- This is because sequencing methods only work on small sections
What was the Human Genome Project?
A project to map the entire sequence of the human genome.
What is a proteome?
All the proteins produced by an organism.
How and why does sequencing the genome of simple organisms help work out their proteome?
They don’t have much non-coding DNA, so the proteome can easily be determined.
When is proteomics useful?
Medical research (e.g. when identifying the antigens on the surface of disease-causing bacteria for vaccine creation)
Why is it hard to work out the proteome from the genome of complex organisms?
- They contain large sections of non-coding DNA and regulatory genes
- These do not code for particular proteins, so it is difficult to translate the genome into a proteome
How many human proteins have been identified so far?
30,000
Give some ways in which sequencing technologies are advancing.
- Automation
- Cost efficiency
- Large scale
Give an example of a development in DNA sequencing.
Pyrosequencing -> Allows 400 million bass to be sequenced in a 10 hour period
What does recombinant DNA technology involve?
Transferring a fragment of DNA from one organism to another.
How does recombinant DNA technology work and why?
- Genetic code is universal and transcription and translation mechanisms are similar too
- So DNA transferred to a different organism can be used to produce the same proteins (even in a different species)
What are organisms that contain transferred DNA called?
Transgenic organisms
Before DNA is inserted into another organism, what must first be done?
DNA fragment must be obtained containing the target gene
What are the 3 ways of producing gene fragments?
1) Reverse transcriptase
2) Restriction endonucleases
3) Gene machine
Describe how reverse transcriptase can be used to make a DNA fragment containing the target gene.
- mRNA is isolated from cells.
- Free DNA nucleotides and reverse transcriptase are added.
- The mRNA molecules can be used as templates for reverse transcriptase to make complementary DNA (cDNA).
What is the advantage of using reverse transcriptase to make DNA fragments containing a target gene?
mRNA created by the target gene is often more abundant than the two copies of the gene itself, so it is easier to obtain
What enzyme can be used to make a complementary DNA strand from mRNA?
Reverse transcriptase
What is the name for DNA produced by reverse transcriptase acting on mRNA?
Complementary DNA (cDNA)
Describe how restriction endonucleases can be used to make a DNA fragment containing the target gene.
- Some genes have a palindromic sequence of nucleotides either side. These are called recognition sequences.
- Restriction endonuclease is chosen that has an active site complementary to the recognition sequences.
- The sample is incubated with the restriction endonuclease, which causes the enzymes to cut the DNA via a hydrolysis reaction.
- This leaves the DNA segment with sticky ends.
What is the name for the place where a restriction endonuclease binds?
Recognition sequence
What makes a recognition sequence characteristic?
Have a palindromic sequence of nucleotides (i.e. with antiparallel base pairs)
Are there multiple different restriction endonucleases?
Yes, each one cuts at a different base sequence.
What type of reaction is a restriction endonuclease cutting DNA?
Hydrolysis
What are sticky ends?
- Small tails of unpaired bases at each end of a DNA fragment
- Created by restriction enzymes.
What are sticky ends used for?
Annealing the DNA fragment to another piece of DNA with sticky ends of complementary sequences.
What is a gene machine?
A technology used to synthesise DNA fragments from scratch without the need for a pre-existing template.
What is the advantage of using the gene machine to produce DNA fragments?
- Pre-existing template is not required
* DNA sequence does not have to exist naturally
Describe how a gene machine can be used to produce a DNA fragment with a target gene.
- DNA fragment is taken from a database or designed
- First nucleotide in the sequence is fixed to a support (e.g. a bead)
- Nucleotides are added step by step in the correct order.
- This involves protecting groups that make sure nucleotides are joined at the right points (to avoid unwanted branching)
- Short sections of DNA called oligonucleotides (about 20 based long) are made.
- These are broken off from the uppity and protecting groups, then the oligonucleotides are joined together.
What are protecting groups?
Groups added to a gene machine to prevent nucleotides from joining at the wrong points (which prevents unwanted branching).
What are oligonucleotides?
- Short sections of DNA
- About 20 nucleotides long
- Joined to make a full DNA fragment in a gene machine
What is amplification?
Producing multiple copies of a section of DNA.
Why is DNA amplified?
To make sure there is enough DNA to work with.
What are the two types of DNA amplification?
- In vivo cloning
* In vitro cloning
What are the stages of in vivo cloning?
1) Insertion of DNA fragment into a vector
2) Vector inserts DNA into host cells
3) Identifying cloned cells
What is a vector?
Something that is used to transfer DNA into a cell.
What are some examples of vector that can be used in in vivo cloning?
- Plasmids
* Bacterioohages (viruses)
In in vivo cloning, describe how the DNA fragment is inserted into a vector.
1) DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment. So the sticky ends are complementary.
2) The vector DNA and DNA fragment are mixed together with DNA ligase, which joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This is called ligation.
3) The new combination is called recombinant DNA.
What enzyme is used when joining a DNA fragment with a vector?
DNA ligase
What is recombinant DNA?
DNA which has been combined from multiple sources.
In in vivo cloning, describe how the vector transfers the DNA fragment into host cells.
1) If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
2) If a bacteriophage vector is used, the bacteriophage will infect the host bacterium by injecting its DNA into it. The recombinant DNA then integrates into the bacterial DNA.
How is a host cell persuaded to take in a plasmid vector containing recombinant DNA?
- The host bacterial cells are placed in ice-cold calcium chloride solution -> This makes their cell walls more permeable
- Plasmids are added
- The mixture is heat-shocked -> This encourages the cells to take in the plasmids
What solution is used to make the cell walls of bacterial cells more permeable so that they take up plasmids more easily?
Calcium chloride
What is heat-shock? Include temperature and time.
Heating to 42°C for 1-2 minutes
How can bacteriophages act as vectors?
They insert their DNA into the host.
What is the term for host cells that take up vectors containing recombinant DNA?
Transformed
In in vitro cloning, how are transformed host cells identified?
1) Marker genes are inserted into vetoes at the same time as the gene to be cloned.
2) Host cells are grown on agar plates.
3) The marker gene can code for antibiotic resistance and the host cells are grown on an agar plate containing the antibiotic, so only transformed cells that have the marker will survive and grow. Or it can code for fluorescence, so transformed cells will fluoresce.
4) Identified transformed cells are allowed to grow more, producing lots of copies of the cloned gene.
What genes can be used as marker genes?
- Antibiotic resistance
* Fluorescence
Describe in detail in vivo cloning.
1) DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment. So the sticky ends are complementary.
2) The vector DNA and DNA fragment are mixed together with DNA ligase, which joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This is called ligation.
3) The new combination is called recombinant DNA.
4) If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
5) If a bacteriophage vector is used, the bacteriophage will infect the host bacterium by injecting its DNA into it. The recombinant DNA then integrates into the bacterial DNA.
6) Marker genes are inserted into vetoes at the same time as the gene to be cloned.
7) Host cells are grown on agar plates.
8) The marker gene can code for antibiotic resistance and the host cells are grown on an agar plate containing the antibiotic, so only transformed cells that have the marker will survive and grow. Or it can code for fluorescence, so transformed cells will fluoresce.
4) Identified transformed cells are allowed to grow more, producing lots of copies of the cloned gene.
How can you ensure that proteins are produced from a DNA fragment after in vivo cloning?
The vector must contain specific promoter and terminator regions.
What are promoter and terminator regions?
- Promoter regions -> DNA sequences that tell the RNA polymerase when to start producing mRNA
- Terminator regions -> DNA sequences that tell RNA polymerase when to stop producing mRNA
How can you ensure that transformed host cells in in vivo cloning contain specific promoter and terminator regions?
• They may be present in the vector DNA
OR
• They may have to be added in along with the target DNA fragment
What process does in vitro cloning use?
Polymerase Chain Reaction (PCR)
Describe the process of in vitro cloning (PCR).
1) DNA fragment, free nucleotides, primers and DNA polymerase are mixed.
2) DNA is heated to 95°C -> To break the H-bonds between the two strands.
3) Mixture is then cooled to between 50°C and 65°C -> Primers can anneal to the strands.
4) Mixture is heated to 72°C -> DNA polymerase can work
5) The DNA polymerase forms a complementary strand due to complementary base pairing.
6) Two new copies of the fragment are formed and the cycle is complete.
7) The cycle starts again.
What are primers?
Short pieces of DNA that allow DNA polymerase to join and start DNA synthesis.
What enzyme is involved in in vitro cloning?
DNA polymerase
What temperatures are used in in vitro cloning?
- Heating to 95°C
- Cooling to 50-65°C
- Heating to 72°C
What happens to the number of copies of the DNA fragment in each cycle of in vitro cloning?
It doubles.
What enzymes are used in in vivo and in vitro cloning?
- In vivo -> Restriction endonuclease + DNA ligase
* In vitro -> DNA polymerase
What organisms can be transformed using recombinant DNA technology (genetic engineering)?
- Microorganisms
- Plants
- Animals
What is genetic engineering?
When organisms are modified using recombinant DNA technology.
Describe how transformed (genetically engineered) microorganism can be produced.
Using the same technology as in vivo cloning.
Describe how transformed (genetically engineered) plants can be produced.
- A desirable gene is inserted into a plasmid
- This is added to a bacterium and the bacterium is used as a vector to get the gene into the plant cells
- If the right promoter region has been added along with the gene, the transformed cells will be able to produce the desired protein
Describe how transformed (genetically engineered) animals can be produced.
- Desirable gene is inserted into an early animal embryo or into the egg cells of the female
- This means that all of the body cells of the resulting animal contain the desirable gene
When genetically engineering an organism, how can you control which cells produce the desired protein?
By using promoter regions that are only activated in specific cell types.
In what areas can recombinant DNA technologies benefit humans?
1) Agriculture
2) Industry
3) Medicine
How can recombinant DNA technologies be used to benefit humans in agriculture?
Agricultural crops can be transformed to:
• Give higher yields
• Be more nutritious
• Have better pest resistance
e.g. Golden Rice
How can recombinant DNA technologies be used to benefit humans in industry?
Biological catalysts can be produced using transformed organisms.
e.g. Chymosin in cheese-making
How can recombinant DNA technologies be used to benefit humans in medicine?
Drugs and vaccines can be produced by transformed organisms.
e.g. Insulin
Into what categories do issues associated with recombinant DNA technology fall?
- Ethical
- Financial
- Social
What are the issues with the use of recombinant DNA technologies in agriculture?
- Monoculture -> Makes the whole crop vulnerable to the same disease
- Superweeds -> If transformed crops interbreed with wild plants, there could be an uncontrolled spread of recombinant DNA with unknown consequences. This could include superweeds, which are resistant to herbicides.
- Contamination of organic crops (so they are no longer legally organic)
What are the issues with the use of recombinant DNA technologies in industry?
- A few large biotechnology companies might force smaller companies out of business
- Without proper labelling, people might be concerned about what they are eating
- Some consumer markets won’t import GM crops, which can cause economic loss to producers
What are the issues with the use of recombinant DNA technologies in medicine?
- Companies who own genetic engineering technologies may limit the use of technologies that could be saving lives
- Ethical issues such as designer babies are a possibility, although it is currently illegal
What are some ownership issues of recombinant DNA technologies?
- There is debate about who owns human genetic material after it has been removed from the body - the donor or the researcher.
- Some large corporations own patents to particular seeds. If non-GM crops are contaminated with this, farmer can be sued for breaching patent laws.
How do humanitarians believe recombinant DNA technologies will benefit people?
- Agricultural crops could be produced that help reduce the risk of famine and malnutrition
- Transformed crops could be used to produce useful pharmaceutical products (e.g. vaccines), which could make drugs more available to more people
- Medicines could be produced more cheaply, so more people can afford them
- Potential for gene therapy to treatment human diseases
What is gene therapy?
Altering the defective genes inside cells to treat genetic disorders.
How can you use gene therapy when a disorder is caused by two mutated recessive alleles?
Add a working dominant allele.
How can you use gene therapy when a disorder is caused by a mutated dominant allele?
“Silence” the dominant allele (e.g. by sticking a section of DNA in the middle so it doesn’t work anymore)
How is gene therapy carried out?
A vector is used to insert an allele into cells (like in recombinant DNA technologies).
What are the two types of gene therapy?
1) Somatic therapy
2) Germ line therapy
What is somatic gene therapy?
- Altering the alleles in body cells, particularly those most affected by the disorder.
- It does not affect the sex cells, so the offspring could inherit the disease.
What is germ line therapy?
- Altering the alleles in sex cells.
* This means that any cell of any offspring produced from these cells will not suffer from the disease.
Is germ line therapy in humans legal?
No
What are DNA probes?
- Short strands of DNA with a specific base sequence that is complementary to a base sequence of a target allele
- It has a label attached to it for detection
What are DNA probes to identify?
Specific alleles of genes to see if a person’s DNA contains a mutated allele that causes a genetic disorder.
What are the two most common type of label on a DNA probe and how are they detected?
- Radioactive -> Detected using X-ray film
* Fluorescent -> Detected using UV light
Describe how a gene probe works to see if a sample has a given allele in it.
- A sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresis
- The separates DNA fragments are then transferred into a nylon membrane and incubated with the fluorescently labelled DNA probe
- If the allele is present, the DNA probe will bind to it
- The membrane is then exposed to UV light and if the allele is present, there will be a fluorescent band
(NOTE: Ask the teacher why you bother to cut up the DNA)
What is a DNA microarray?
A glass slide with microscopic spots of different DNA probes attached to it in rows
Describe how a DNA microarray is used to find which alleles a sample of DNA contains.
- A DNA array is a glass slide with microscopic spots of different DNA probes attached to it in rows
- A sample of fluorescently labelled human DNA is washed over the array
- If the labelled human DNA contains any DNA sequences that match any of the probes, it will stick to the array
- The array is then washed to remove any DNA that hasn’t stuck
- The array is then visualised under UV light -> Any labelled DNA attached to a probe will show up
- Any spot that fluoresces means that the person’s DNA contains that specific allele
(See diagram pg 218 of revision guide)
How can you produce a DNA probe?
- Sequence the allele you want to screen for
* Use PCR to produce multiple complementary copies of part of the allele (these are the probes)
What things can DNA probes be used for?
1) Identifying inherited conditions
2) Determining how a patient will respond to specific drugs
3) Health risks
Give an example of how DNA probes can be used to identify inherited conditions.
- Huntington’s disease
* Only starts to show symptoms between the age of 30 and 50, but it CNS be screened for before then
Give an example of how DNA probes can be used to determine how a patient will respond to certain drugs.
- Breast cancer may be caused by a mutation in the HER2 proto-oncogene
- Screening for this can help determine whether Herceptin will be a useful treatment or not
Give an example of how DNA probes can be used to identify health risks.
- Inheriting particular mutated alleles increases your risk of developing certain cancers.
- Screening for these can help inform life choices to prevent these cancers.
What is genetic counselling?
Advising patients and their relatives about the risks of genetic disorders
What does genetic counselling involve?
- Advising people about screening
- Explaining the results of screening
- Explaining possible prevention or treatment options
Remember to revise the two examples on genetic counselling on pg 219 of revision guide.
Do it.
What is personalised medicine?
Tailoring treatment plans to a person’s DNA.
What are the parts of DNA that are non-coding made up of?
Variable Number Tandem Repeats (VNTRs)
What are VNTRs?
Base sequences that don’t code for proteins and repeat next to each other over and over.
Are VNTRs the same in all humans?
No, the number of times a sequence repeats different between individuals.
e.g. A four nucleotide sequence might be repeated 12 times in one person (= 48 nucleotides) but 16 times in another person (= 64 nucleotides).
Can a given sequence in VNTRs be repeated at multiple points in a genome?
Yes
Describe the principle of why VNTRs can be used in genetic fingerprinting.
- VNTRs differ in length between people
- Since a sequence can be repeated at multiple points along the genome, it’s highly unlikely that two people will have the same length of a VNTR at every point in the genome
What is genetic fingerprinting?
Comparing the lengths of VNTRs between individuals in order to identify them.
Describe how genetic fingerprinting can be carried out on a sample of DNA.
1) Sample of DNA is obtained (from blood, saliva, etc.)
2) PCR is used to make many copies of the areas of DNA that contain the VNTRs -> Primers are used to designed to bind either side of these repeats and so each whole repeat is amplified
3) You now have multiple DNA fragments where the length corresponds to the number of VNTRs each person has at each locus
4) Fluorescent tag is added to all the DNA fragments so they can be viewed under UV light
5) The DNA fragments undergo electrophoresis to separate them out by size
6) The DNA fragments are viewed as bands under UV light -> This is the genetic fingerprint
(See pg 220 of revision guide)
Remember to practise writing out how a genetic fingerprint is prepared.
Pg 220 of revision guide
What are the main uses of genetic fingerprinting?
- Determining relationships
- Determining genetic variability within a population
- Forensic science
- Medical diagnosis
- Animal and plant breeding
How can genetic fingerprinting be used in determining relationships?
- Compare the genetic fingerprints of a child and possible parent
- The more bands match, the more closely related the two people are
- Every single one of the bands on a child’s fingerprint should be accounted for by a band on either the mother’s or father’s genetic fingerprint
How can genetic fingerprints be used to calculate genetic variability within a population?
- Look at a given locus in the genome
- Compare how the number of repeats there varies within the population
- Repeat at multiple loci
How can genetic fingerprinting be used in forensic science?
- Samples of DNA collected from crime scenes can be compared to DNA samples from suspects.
- This is done by comparing the genetic fingerprints of the sample form the scene and each of the suspects
- The one where the bands all match is the owner of the DNA from the crime scene
In medical diagnosis, what can a genetic fingerprint refer to?
A unique pattern of several alleles.
Describe how genetic fingerprinting can be used in medical diagnosis.
- It can be used to diagnose genetic disorders and cancer.
- This is useful when scientists do not known which allele of which gene is responsible for a disease or where several mutations could have caused the disease
- The reason for this is that the fingerprint identifies broader areas of the genome that are abnormal
Describe how genetic fingerprints can be used in preimplantation genetic haplotyping (PGH).
- PGH is where embryos in IVF are screened before implantation
- Faulty regions of the parents’ DNA are used to make genetic fingerprints
- These can them be compared to the genetic fingerprint of the embryo to see which regions have been inherited (i.e. whether a faulty region of the genome has been inherited)
Describe how genetic fingerprints can be used in diagnosing tumours.
- Conventional methods of identifying tumours only show physical differences between tumours
- However, now, the genetic fingerprint of a tumour can be compared to the fingers of known tumour types
- If there’s a match, the tumour can be specifically diagnosed and the treatment can be targeted to that specific type
Describe how genetic fingerprinting can be used in animal and plant breeding.
It is used to prevent inbreeding, which has an increased risk of genetic disorders.
