Flashcards in 16. DNA Technology Deck (79):
Explain how enzymes and vectors can be used to isolate genes and insert them into another organism.
-Cut DNA at specific base sequences
-Same (restriction) enzyme also cuts DNA into which gene is inserted/plasmid/virus/Agrobacterium
-Joins two pieces of DNA together/ forms recombinant DNA
-Vector needed to insert DNA into host/plasmid/second organism
-Correct ref to sticky ends; reverse transcriptase; mRNA-->DNA
Describe the similarities and differences between replication and transcription.
-H bonds break/ DNA unzips
-Between complimentary bases
-Complimentary nucleotides/ bases added/ DNA acts as template
-Same, correctly named, enzymes eg polymerase
-Uracil/ thymine used
-All copied or only section copied respectively
-One strand used in transcription, two in replication
-Enzymes that are different, correctly named
Describe how the new protein is made once the gene has been inserted into the cell.
-Unwinding/ unzipping DNA
-Involving breaking hydrogen bonds
-Assembly of mRNA nucleotides
-Complementary base pairing
-Role of polymerase enzymes
-mRNA enters ribosome
-Specific tRNA molecule associated with specific amino acid
-Formation of peptide bonds
-Specific role of ATP/ energy
-Reference to gene switched on
What is DNA from two different organisms combined called?
What are the five stages involved in making a protein using the DNA technology of gene transfer and cloning?
1) isolation - DNA fragments that have the gene for the desired protein are isolated
2) insertion - fragment of DNA inserted into a vector
3) transformation - transfer of DNA into suitable host cells
4) identification - see if host cells have successfully taken up the gene by using GENE MARKERS
5) growth/cloning - cloning of population of host cells
What is a retrovirus and in what form is its genetic material? What enzyme can we use?
A retrovirus is a group of viruses, a notable example is HIV, which have their genetic information stored as RNA.
Therefore we can use reverse transcriptase to form DNA from RNA
Give an example to explain how a specific protein can be harvested using reverse transcriptase
Cell that readily produces the protein is selected (eg if you want insulin then use beta cells in the pancreas)
They will produce lots of the relevant mRNA which is extracted
Reverse transcriptase is used to make the mRNA into DNA (called cDNA, complementary DNA)
DNA polymerase is used to build up complementary nucleotides on the cDNA template- the double strand of DNA is the required gene
What is the DNA formed from RNA using reverse transcriptase called?
cDNA, complimentary DNA
What does a restriction endonuclease do?
It recognises DNA at a specific sequence of bases and cuts it.
These sequences occur in the DNA of all species of organisms, but not in the same places!
What is a natural use of restriction endonucleases?
Some virus's inject their DNA into bacteria in order to take over the cell. Some bacteria defend themselves by producing enzymes that cut up the viral DNA- restriction endonucleases!
What are blunt ends and what are sticky ends?
Blunt ends occur when a restriction endonuclease cuts at a recognition site and cuts between two opposite base pairs.
Sticky ends occur when restriction endonucleases cut the DNA in a staggered fashion, leaving each end of the strand of DNA with exposed, unpaired bases.
What is a typical feature of sticky ends when cut by restriction endonucleases?
Sometimes the recognition sequence is a, for example, a six base pair palindromic sequence
Palindromic sequences are typical because
What are the two ways of cloning fragments of DNA that have been obtained?
-in vivo- transferring the fragments into a host cell using a vector
-in vitro- using the polymerase chain reaction
What is the importance of sticky ends?
It means DNA can be inserted into, for example, a plasmid because the same restriction endonuclease is used to cut both DNAs (the recognition site is the same as you use the same R.E) so the nucleotides of one sticky end are complimentary to the place in the host DNA where you want to insert it
How is the sugar phosphate backbone joined once the sticky ends have formed complimentary base pairing?
What is a plasmid and why is it useful?
They are circular lengths of DNA, separate from the main bacterial DNA
They almost always contain genes for antibiotic resistance so restriction endonucleases can be used at one of these genes to brisk the plasmid loop
They are useful as vectors
What is a vector?
Something that carries DNA into a host cell
Describe the process of transformation (getting a plasmid with the gene into the host cell, bacteria)
Mixture of plasmids and bacterial cells are mixed together in a medium containing calcium ions and heated.
Calcium ions and increased temperature make the bacteria permeable, allowing the plasmids to pass through the cell membranes.
After the process of transformation, why do not all of the bacteria contain the DNA fragments?
-only a few bacterial cells (as few as one percent) take up the plasmids when they are mixed together
-some plasmids will have closed up again without incorporating the DNA fragment
How do you identify which bacterial cells have taken up the plasmid with the inserted gene?
-use replica plating (antibiotic resistance gene markers)
-put bacterial colonies on plates with different antibiotics that you know the plasmid with the inserted gene has resistance against
-colonies that grow on the plates with the antibiotic have the plasmid because they have antibiotic resistance, those that die are the ones that did not absorb a plasmid
How do you identify which bacterial cells have taken up plasmids
-with no extra DNA
-with the gene successfully inserted?
>antibiotic resistance marker
Describe how antibiotic resistance markers work. (Plasmids that have taken up genes will have non functional tetracycline resistance gene)
-use replica plating
-bacterial cells that survived the ampicillin antibiotic are known to have taken up the plasmid
-these cells are cultured by spreading them thinly on nutrient agar plates
-each separate cell will grow into a genetically identical colony
-tiny sample of each colony is transferred onto a second (replica) plate in the same position as the original colonies on the plate
-second replica plate contains the second antibiotic, tetracycline, against which the antibiotic resistance gene will have been made useless if the new gene has been taken up
-colonies killed by the antibiotic must be the ones that have taken up the required gene- these colonies therefore are made up of bacteria that has been transformed (has been genetically modified)
Describe how fluorescent markers are used to identify whether or not the plasmid has been taken up by host.
-transfer jellyfish dna for the production of a green fluorescent protein (GFP) into the plasmid
- the gene to be cloned is put in the middle of the GFP gene, meaning that those cells that have taken up the gene will not flouresce because the gene will have been inserted into the middle of the GFP gene, making it non functional
-you can therefore find the cells that have the desired gene by looking under a microscope and seeing which cells do not fluoresce.
Describe how enzyme markers are used to find out if cells have taken up the plasmid with the desired gene. give an example.
-one example of a gene marker is lactase which turns a particular colourless substrate blue.
-required gene is transplanted into the gene that makes lactase
-if the gene is inserted correctly then no blue colour will be formed as lactase cannot be produces as the gene has broken the lactase gene
-blue colour will appear if the plasmid taken up has not had the desired DNA inserted into it
What five things does the polymerase chain reaction require?
-the dna fragment to be copied
-dna polymerase (obtained from bacteria in hot springs so it is not denatured by the heat)
-primers- short sequences of nucleotides with sets of bases complimentary to one end of each of the two dna fragments
-nucleotides - which contain each of the 4 bases found in DNA
-thermocycler- a machine that varies temperature precisely over time
Describe the series of events that occur in the polymerase chain reaction.
-dna strands heated to SEPARATE the two strands (95degrees celsius)
-PRIMERS are annealed- the mixture is cooled to 55 degrees celsius which causes the primers to anneal. The primers provide the starting sequences for DNA polymerase
>primers provide the starting sequence for dna polymerase to begin dna copying because dna polmerase can only attach nucleotides to the end of an existing chain
>primers also prevent the two separate dna strands from rejoining
-SYNTHESIS of dna- temperature increased to 72 degrees so that dna polymerase can add complementary nucleotides to each of the separate dna strands
What are the advantages of in vitro gene cloning?
-it is extremely rapid and can be used even when there is very little genetic material available
-does not require living cells- no complex culturing techniques which require lots of time and effort
What are the advantages of in vivo cloning?
- useful when we want to introduce a gene into another organism
-involves almost no risk of contamination- contaminant dna will not be inserted into the plasmid because as they are cut with a certain restriction endonuclease, the sticky ends are complimentary with the desired gene
-it is very accurate- mutations are rare however in in vitro cloning errors in copying dna can be copied may times
-it cuts out specific genes- not just copies of whole dna samples
-produces large quantities of transformed bacteria that can be used to produce large quantities of gene products eg insulin.
How can genetic modification benefit humans?
-increasing yield from animals or plants
-improving nutrient content of food
-introducing resistance to disease and pests
-making crop plants tolerant to herbicides
-developing tolerance to environmental conditions eg extreme temperatures and drought
-producing medicines for treating disease
Give the names of the three main groups of the substances produced using genetically modified bacteria, with examples of each.
-antibiotics- although GM has not substantially improved he quality of the antibiotics, using bacteria means that a greater quantity of antibiotic can be produced at a greater rate
-hormones- bacteria can be GM'd to produce insulin where previously it was harvested from pigs and cows which would result in rejection of insulin from the patients body and the ethical issues of killing the animals
-enzymes- enzymes such as amylase are manufactured by genetically modified bacteria to be used for improve the flavour of cheese and tenderise meat etc...
Give five examples of genetically modified plants.
1) genetically modified tomatoes- gene inserted that gas a complimentary base sequence to the gene that produced the enzyme that causes the tomatoes to be soft so the mRNA of the inserted gene is complementary to the mRNA for the original gene. This means that the two combine to form a double strand, preventing the mRNA of the original gene from being translated. Therefore the tomatoes still develop flavour without the problem of harvesting and transporting soft tomatoes
2) herbicide resistant crops- crops unaffected by herbicide but the weeds which compete with the crop for light, minerals and water are killed
3) disease resistant crops- resistance to specific diseases eg. GM rice can withstand infection from a particular virus
4) pest resistant crops-eg maize can have a gene added that causes it to produce a certain toxin which kills the insects that eat the maize but is harmless to other animals, including humans
5)plants that produce plastics- currently being explored. Plants can be GM'd to have the metabolic pathways necessary to make the raw material for plastic production
Give examples of genetically modified animals.
Fish and sheep can have a growth hormone added so that, in the case of salmon, they can grow 30 times larger at 10 times the usual rate
Insert a gene for resistance for a particular disease from one animal to another, making the second animal resistant to the disease
GM milk producing animals to produce a particular protein in their milk which can then be easily harvested
How can goats be genetically modified to produce something in their milk, using an example
-anti thrombin can be produced from goats milk to treat people prone to blood clots who cannot produce their own antithrombin
-mature eggs removed from female goats and fertilised by sperm
-normal gene for thrombin production from a human is added to the fertilised eggs along side the gene that codes for proteins in goats milk
-genetically transformed eggs are implanted into female goats
-kids with the antithrombin gene are cross bred
-milk from goat herd is rich in antithrombin
-antithrombin is given to humans unable to manufacture their own
What is cystic fibrosis And what causes it?
Caused by a recessive allele in which there is a deletion of AAA in the CFTR gene(deletion mutation) (cystic fibrosis trans-membrane-conductance regulator) so the protein is missing one amino acid which makes it unable to transport chloride ions out of epithelial cells, with water following by osmosis so therefore epithelial membranes are not kept moist so the mucus is too thick and viscous.
What are the symptoms of cystic fibrosis?
-mucus congestion in the lungs, leading to a higher risk of infection as bacteria is trapped in the mucus
-breathing difficulties and less efficient gas exchange
-accumulation of thick mucus in the pancreatic ducts, preventing pancreatic enzymes from reaching the duodenum and leading to the formation of fibrous cysts
-accumulation of mucus in sperm ducts in males, possibly leading to infertility
What are the two ways in which cystic fibrosis can be treated using gene therapy?
-gene replacement- defective gene is replaced with a healthy gene
-gene supplementation- in which one or more copies of the healthy gene are added along side the defective gene - as the genes added have dominant alleles so the effects of the recessive alleles are masked
Describe germ-line gene therapy and somatic-cell gene therapy
Germ-line gene therapy- replacing or supplementing the defective gene in the fertilised egg, ensuring that all cells of the organism will develop normally, as will all the cells of their offspring-more permanent solution
Somatic cell gene therapy- just targets the affected tissues and is not present in the sperm or egg cells so is not passed on to future generations. However this needs to be repeated as often as every two days because the cells of the affected tissues die and are replaced fairly regularly
Describe how a virus is used to deliver cloned CFTR genes
Adenoviruses inject their DNA into cells. This is how adenoviruses are modified so that they can serve as a vector for the normal CFTR gene:
-adenovirus made harmless by interfering with a gene that is involved in their replication
-adenoviruses grown in epithelial cells in the lab along with plasmids that have the normal CFTR gene inserted
-CFTR gene becomes incorporated into the DNA of the adenovirus
-adenoviruses isolated from the epithelial cells and purified
-adenoviruses with CFTR gene introduced into patients via nostril
-adenoviruses inject their DNA including the normal CFTR gene into the epithelial cells of the lungs
Describe how liposomes can be used to treat cystic fibrosis in gene therapy
-CFTR genes isolated from healthy humans and inserted into bacterial plasmid vectors
-plasmids reintroduced into their bacterial host cells with gene markers to identify which bacteria have successfully taken up the plasmid with the CFTR gene
-bacteria cloned to produce multiple copies of the plasmids with CFTR gene
-plasmids extracted from bacteria and wrapped in lipid molecules to form a liposome
-liposomes sprayed into nostrils
-liposomes pass through phospholipid bilayer into epithelial cells
Why are adenoviruses and liposomes not always successful?
-adenoviruses may cause infections
-patients may develop immunity to adenoviruses
-liposomes aerosol may not be fine enough to pass through the tiny bronchioles in the lungs
-even if CFTR gene is successfully delivered, very few are expressed
How severe combined immunodeficiency (SCID) treated using gene therapy? (SCID is caused by a defect in the gene that codes for ADA enzyme which breaks down the toxins that would otherwise kill white blood cells)
-normal ADA gene isolated from healthy human tissue using restriction endonucleases
-ADA gene inserted into a retrovirus
-retroviruses grown with host cells in the lab to increase their number
-retrovirus mixed in with the patients T cells
-retroviruses inject a copy of the normal ADA gene into the T cells
-T cells are reintroduced into the patients blood to provide the genetic code needed to make ADA
Why does gene therapy, so far, only have limited success?
-the effect is short lived - somatic cells are not passed on to daughter cells so repeat treatments are necessary
-it can induce an immune response so often the treatment is rejected
-genes are not always expressed
-it is not effective at treating conditions that arise in more that one gene
-viral vectors sometimes present problems- could lead to toxic, inflammatory and immune responses in recipients/ 'disabled' viruses may recover the ability to cause disease once inside the patient
What is a DNA probe and what are the two most common types?
It is a short single stranded section of DNA that has some kind of label attached that makes it easily identifiable.
-radioactively labelled probes made of nucleotides with isotope P-32 which is identified using a photographic plate that is exposed by radioactivity
-fluorescently labelled probes which emit fluorescent light under certain conditions
How is a DNA probe used to identify particular genes?
Probe has a base sequence complementary to the portion of the DNA sequence that makes up part of the gene whose position we want to find
DNA that us being tested is treated to separate its two strands
Separated strands mixed with a probe which binds to the complementary bases on one of the strands -this is called DNA HYBRIDISATION
site at which the probe binds is identified using the radioactivity or fluorescence that the probe emits
Describe what is put into the four test tubes in Sanger sequencing.
-single stranded fragments of DNA that is to be sequenced (acts as a template for the synthesis of its complementary strand
-mixture of nucleotides with A, T, C and G
-Small quantity of the four terminator nucleotides
-primer to start the process of DNA synthesis (radioactively or fluorescently labelled primers used
-DNA polymerase to catalyse DNA synthesis
What happens in Sanger sequencing?
The radioactively labelled terminator nucleotides are bound to the template randomly so the sequence could be terminated anywhere so the DNA fragments will be of varying length. They are then run on an electrophoresis plate which separates the fragments by size and you can then read the code of the DNA by reading the terminator nucleotides in order of length!
How does gel electrophoresis work?
DNA fragments are placed onto an agar gel and a voltage is applied across it, causing the negatively charged DNA fragments to move across the gel to the positive electrode. Due to the resistance of the gel, the smaller DNA fragments will travel further than the larger ones in a given time.
How does restriction mapping work?
Different restriction endonucleases are used to cut a plasmid at their different restriction sites and this is run on an electrophoresis plate. From the length of the fragments you can determine the distance between the recognition sites
What is genetic screening?
A genetic technique to determine whether an unborn child will have a genetic disorder
What is a gene probe?
-A short length/ stand of DNA
-Short strand/ up to 20 bases long
-With base sequence that is complementary to part of target gene
-Which is fluorescently or radioactively labelled
Give two ways in which the information obtained from using a gene probe would be useful to a doctor who is counselling someone with a family history of cancer.
-Identify carrier of cancer gene
-Identify which cancer gene is present
-Identify most effective therapy
Describe how PCR is carried out.
1 DNA heated to 90 to 95°C;
2 strands separate;
3 cooled / to temperature below 70°C
4 primers bind;
5 nucleotides attach;
6 by complementary base pairing;
7 temperature 70 - 75°C;
8 DNA polymerase joins nucleotides together;
9 cycle repeated;
About 20% of DNA produced by PCR is copied inaccurately . Suggest and explain why it is not safe to use PCR to clone the CFTR gene for use in treating cystic fibrosis.
-Percentage risk too high for human applications
-Different tRNA/ tRNA brings wrong amino acid
-Structure of protein synthesised unknown/sequence of amino acids changed/ incorrect shape
-Produce harmful/ toxic protein
-Protein non functional/chloride ions not transported
Some human DNA was cut into separate pieces using a restriction enzyme
which produced a staggered cut. A scientist wanted to insert these pieces of DNA
into plasmids and used the same restriction enzyme to cut the plasmids. Explain
why the pieces of human DNA would be able to join to the cut DNA of the plasmids.
-Sticky ends/ description
-Reference to complementary base pairing
After using restriction enzymes to cut a plasmid and the target DNA, which enzyme must the scientist add to form recombinant plasmids?
A plasmid may be used as a vector. Explain what is meant by a vector in this context.
-Into cell/ other organism/ host
(2 marks) Molecular biologists often use plasmids which contain antibiotic resistance genes. Explain the reason for this.
-Act as a gene marker
-Allows detection of cells containing plasmid/ DNA
-Reference to growing bacteria on antibiotic
What is meant by a gene?
-Length of DNA
-That codes for a single protein/ polypeptide
In a particular PCR, two different primers are added.
-Why are primers required?
-Suggest why two different primers are required.
-To allow DNA polymerase to attach/ start addition of nucleotides/ mark start and end of sequence to be copied/ prevents strands from rejoining.
-Because the sequences at the ends of the target sequence are different/ one is at the beginning and one is at the end
From starting with a single strand of DNA, the polymerase chain reaction was allowed to go through three complete cycles. At the end of the three cycles, how many molecules of DNA will be produced?
Explain the purpose of attaching a gene for a green fluorescent protein to the target DNA from a healythy human when genetically modifying goats so they produce a factor IX, for people who do not produce factor IX, in their milk.
-Act as a gene marker
-Shows that the human gene has been taken up/ expressed
-Only implant eggs that fluoresce/ contain the jellyfish gene
It is important that scientists still report results from failed attempts at producing transgenic animals. Explain why.
-Saves time/ money for others
-Same work is not repeated/methods can be compared/ improved/ amended/ same errors not made again
Explain why EcoRI only digests DNA at the specific recognition sequence.
-Shape/ configuration complementary to shape of active site of enzyme
Give two characteristic features of stem cells
-Will replace themselves/ keep dividing/ replicate
The other viral enzyme is called integrase. Integrase inserts the DNA copy anywhere in the DNA
of the host cell. It may even insert the DNA copy in one of the host cell’s genes.
The insertion of the DNA copy in one of the host cell’s genes may cause the cell to
make a non-functional protein. Explain how.
-Alters nucleotide sequence causes frame shift
-Different sequence of amino acids in poly peptide
-Alters tertiary structure
Some children in the trial developed cancer. How might insertion of DNA using integrase have caused cancer?
-Affects tumour suppressor gene
-Inactivates tumour suppressor gene
-Rate of cell division increased/tumour cells continue to divide
How can someone be tested for many different genetic disorders simultaneously?
-Fix hundreds of DNA probes in an array (pattern) on a glass slide
-Add a sample of DNA to the array
-Any complementary sequences in the DNA of the donor will bind to one or more probes
What is a tumour suppressor gene?
-Genes that inhibit cell division
How may a tumour form?
-Mutations on both alleles of the tumour suppressor gene must be present to inactivate the tumour suppressor gene and initiate the development of a tumour.
In cases of cancer, what can screening help to detect?
-Oncogene mutations which determine the type of cancer the person has and therefore which treatments will be the most effective.
-Gene changes which predict which patients will be more likely to benefit from certain treatments eg. herceptin (drug) is most effective at treating certain types of breast cancer
-A single cancer cell among millions of normal cells, thus identifying patients at risk of relapse from certain forms of leukaemia
What is a core sequence?
Repetitive sequences of DNA in introns.
What are the five main stages of making a genetic fingerprint? (Even Ducks Sometimes Hate Ducks)
-EXTRACTION- separate DNA from the rest of the cell. If sample of DNA is tiny, numbers can be increased using the polymerase chain reaction.
-DIGESTION- DNA cut into fragments using restriction endonucleases- endonucleases chosen for their ability to cut close to but not within groups of core sequences
-SEPARATION- Separated by size using gel electrophoresis
-HYBRIDISATION- radioactive or fluorescent DNA probes are now used to bind with the core sequence. Probes have a base sequence complementary to the core sequences and binds to them under specific conditions (pH, temperature). The process is carried out with different probes, each of which binds to a different core sequence.
-DEVELOPMENT- X ray film put over the nylon membrane and the film is exposed by the radioactive probes (if fluorescent, the positions can be determined visually). Because the points correspond to the position of DNA fragments separated by gel electrophoresis, a series of bars is revealed. The pattern of bands is unique for all individuals except identical twins.
What is southern blotting?
-A gel electrophoresis is immersed in an alkali to separate the double strands into single strands
-Single strands transferred onto a nylon membrane by a technique called southern blotting:
-Thin nylon membrane is laid over the gel
-Membrane covered with several layers of absorbent paper which draws up the liquid containing the DNA through capillary action
-This transfers the DNA fragments onto the nylon membrane in precisely the same relative positions that they occupied on the gel
-DNA fragments fixed to the membrane using ultraviolet light
What are the uses of DNA fingerprinting?
-See if an individual was involved in a crime- forensic science
-To establish if someone is the genetic father of a child
-Determining genetic variability within a population
Scientists can separate fragments of DNA using electrophoresis. Suggest how they
can use electrophoresis to estimate the number of base pairs in the separated
Give one mark for answer confined to smaller fragments
Give two marks for comparing with distance/speed moved by
fragments of known size/markers / DNA ladder;;
Scientists need to take precautions when they carry out restriction mapping. They
need to make sure that the enzyme they have used has completely digested the DNA.
One check they may carry out is to add the sizes of the fragments together.
How could scientists use this information to show that the DNA has not been
completely digested? Explain your answer.
. Large pieces of DNA present;
2. Add up to more than total length of original DNA / plasmid
plus inserted DNA;
3. Because this would add undigested to total (original)
The jellyfish gene attached to the human Factor IX gene (Stage 2) codes for a protein
that glows green under fluorescent light. Explain the purpose of attaching this gene
1. (Acts as a) marker gene;
2. Shows that the (human)
gene has been taken
3. (Only) implant cells/embryos
that show fluorescence /
contain the jellyfish gene;
Many attempts to produce transgenic animals have failed. Very few live births result
from the many embryos that are implanted.
Suggest one reason why very few live births result from the many embryos that are
1. Mutation / nucleus/
chromosomes/DNA may be
damaged / disrupts genes;
2. May interfere with proteins
3. Embryo/antigens foreign;
4. Embryo is rejected/attacked
by immune system;