Unit 4.5 - Application of reproduction and genetics Flashcards
Goal of the human genome project
To map the human genome
Intended purpose of the human genome and 100k projects
To improve knowledge and understanding of genetic disorders and improve their diagnosis and treatment (more accurate)
What does a diploid cell’s DNA from a human contain?
Every gene that humanity possesses - the whole genome
Genome
The total number of all the genes a species possesses
If every cell in a human has the same genome, how are we different?
They may not contain all of the alleles of the genes
How was the human genome project completed?
Lots of labs worldwide shared their data and different labs worked on different sections of the human genome
Why did genes have to be extracted before they were sequenced for the human genome project?
In the DNA of a cell, there are also introns that are non-coding
What type of sequencing did the human genome project use?
Sanger sequencing
How big are the sections of dna used in Sanger sequencing?
Relatively small sections of dna at a time (usually <1000bps)
How long did the human genome project take?
13 years
Why was the human genome project completed faster than expected?
Since the technology was adapting quickly
How fast could we sequence an entire human genome now?
In a mater of hours - genomes are routinely sequenced
What do we now know as a result of the human genome project?
The sequence of bases in every gene
That there are 20,000 genes in the human genome
Explain in detail the potential benefits of the human genome project
Development of ew and better targeted medical treatments (e.g - cancer used o be treated in the same way in every patient no matter what type of cancer it was. Now individuals are treated depending on their genome, since different people respond differently to treatments depending on their genetic makeup, so targeted treatments can be developed
Increased opportunities for screening for genetic disorders. If we know the normal genome, we can easily identify mutations and anomalies that cause diseases. By knowing the sequence of the allele(s) that causes a genetically determined disease, scientists can determine whether a person will develop a disease.
Better prediction of the effect of drugs
Scientists can look for incidences of mutation in certain genes that may result in genetic disorders
Technique used to sequence the genome
Sanger sequencing
What’s Sanger sequencing used for?
Sequencing the genome
What is Sanger sequencing used for?
To read the order of the nucleotides with every base in every gene
Explain how Sanger sequencing is done
Sanger sequencing makes copies of the gene in the lab using free nucleotides - the DNA is heated so that the strands separate and the bases on free nucleotides match with complementary bases on the target DNA in order to make a copy of the DNA. However, along with the normal nucleotides, you include dideoxynucleotides.
Why is dna heated during Sanger sequencing?
So the the strands separate and the bases on free nucleotides match with complementary bases on the target DNA in order to make a copy of the dna
What’s included along with the normal nucleotides during Sanger sequencing?
Dideoxynucleotides
How does dna replication occur in the usual reactions?
Bases match with complementary bases and a reaction has to happen to form the sugar-phosphate backbone
Which groups react together for complementary bases to join together?
The OH- on the carbon-3 on the deoxyribose reacts with the OH- on the phosphate group in the next nucleotide
What type of reaction joins two bases?
Condensation
What are used instead of deoxyribose in Sanger sequencing?
Dideoxyribose bases
What cannot happen when dideoxyribose is used in Sanger sequencing and why? Explain
In dideoxyribose, there’s no OH, so the bases cannot join since the condensation reaction cannot occur (no loss of OH from the carbon-3 atom)
What happens when bonds can’t form due to a dideoxyribose in Sanger sequencing?
The chain stops growing
What is there also on a dideoxyribose base?
A radioactive label
What happens to the dna sequence when the dideoxyribose is incorporated?
It’s terminated
How come we get different lengths of dna strands in Sanger sequencing?
Since not every, for example, guanine, will have the dideoxyribose sugar. Some will have the normal deoxyribose sugar.
What will the length of the dna strand depend on if guanine is the base with the dideoxyirbose sugar for example?
Depends on where the dideoxyribose guanine is
Can different bases be used with dideoxyribose sugars?
Yes
What do we need to do after Sanger sequencing?
Compare the strands of dna
How do we compare strands of dna after Sanger sequencing?
Run them on an electrophoresis gel
Electrophoresis gel
A slab of agarose gel in a tank with a buffer
How is electrophoresis done?
An electric charge is set up from one end of the gel to the other
The dna samples are then put into wells at one end and the electric charge is run
Where is dna drawn towards during gel electrophoresis and why?
Towards the positive charge in the tank since its a negatively charged molecule since the phosphate groups have a negative charge
How come dna is able to be pulled through the gel during gel electrophoresis?
Since the gel is porous
Describe and explain the relationship between the length of the dna strand and the speed at which it travels through the gel during gel electrophoresis
The longer the dna strand, the slower it travel through the gel since there’s more resistance from the gel. So smaller pieces travel further over a certain amount of time.
Which pieces of dna travel further over a certain amount of time during gel electrophoresis and why?
Smaller pieces
Less resistance from the gel
What happens once dna has been pulled through the gel during gel electrophoresis?
The dna is sorted into bands which contain dna of the same length
Which strands of dna will have travelled furthest during gel electrophoresis?
The smallest pieces
How can we determine the letters of the base sequence using gel electrophoresis?
Wherever it terminated is the letter
Would we be able to actually see the bands during gel electrophoresis?
No - they would actually be invisible in the gel
How do we make the bands from gel electrophoresis visible? Explain
Since the dideoxyribose bases were labelled with radioactive labels, we old be able to place a piece of photographic film on the gel in the dark and the bands would appear on the film due to the radioactivity
How one the human genome project sped up and got more accurate and automated as it went along?
Instead of using the electrophoresis gel, next generation (NGS) was used
Why is NGS better than using gel electrophoresis?
It’s much faster - you could sequence an entire genome in a few hours
How long does it take to sequence an entire endometriosis with next generation sequencing?
A few hours
Explain in detail how next generation sequencing is done
DNA strands are put into a capillary tube
The labels on the terminator bases are coloured instead of radioactive and fluoresce under UV light
There’s a laser at the end of the capillary tube and the terminator bases light up different colours evening on the base
The shortest dna strands are read first since they travelled faster
You eventually get a series of coloured blocks and these odours can be converted into the letters of the base sequence
Which dna strands are read first during next generation sequencing and why?
The shortest ones - they travelled faster
What other species have genome projects also been completed for?
A number of species including chimpanzees and other primates
What has completing genome projects on other species allowed scientists to do? Explain
Look at evolutionary relationships. This provides true phylogenetic classification and can be used to correct mistakes made using classification based on phenotypic characteristics
Consider how to conserve species in the future by targeting which species need particular attention. This is done by looking at dna variation between species (low variation = more endangered and more likely to become extinct)
What is malaria caused by?
A parasitic protist which reproduces inside cells in the body
How is malaria transmitted from person to person (i.e - what is the vector)?
Anopheles Gambiae (a mosquito)
How is the spread of malaria usually treated?
By killing the mosquitos with an insecticide
Why is killing mosquitos with an insecticide not an effective method against malaria anymore?
Rapid evolution of insecticide resistance in the species is hampering attempts to eradicate the disease which is responsible for over a million deaths per year
Malarial parasite and the issue with it
Plasmodium sp.
Developed anti-drug resistance
How is malaria being combatted?
Scientist are working on projects with the aim of sequencing the entire genome of both the malarial parasite plasmodium and the anopheles mosquito
Why are scientists working on projects with the aim of sequencing the entire genome of both the malarial parasite plasmodium and the anopheles mosquito?
For the mosquitos, this will allow us to find out where the mutation is that gives them resistance and target it to make the insecticide more effective
For the parasite, this will allow us to target certain geese with drugs that will treat malaria
This will hopefully eradicate the disease
Name a genetics project that’s currently ongoing
The 100,000 genome project
What can NGS do in a few hours?
Sequence an entire genome
What is NGS allowing scientists to do in the 100k genome project?
To study variation within the human genome amongst 100,000 people in the uk with rare genetic disorders
Their gnomes are looked at to see how their dna sequence changes to cause the rare genetic disorder
Principle aims of the 100k genome project
Study variation within the human genome
Create a new genomic medicine service for the NHS - a database would be made over time to help the genomic medicine service
Enable new medical research to study the potential of new and more effective treatments
Study how best to use genomics in healthcare and how best to interpret the data to help patients
Kick start a uk genomics industry
Genomics
Use DNA sequencing to identify differences in the dna to the usual genome to target treatments towards the individual
How could genomics be used in healthcare?
We could start sequencing genomes at the doctor to develop treatments targeted towards the individual’s genome
What has been produced by both the human genome and 100k projects?
A vast quantity of data and its potential is profound
What’s the issue with the human genome and 100k project?
We do not know how this information might be used in future
What is society struggling to decide when it comes to the human genome and 100k projects?
Where the legal and moral responsibility for this information lies
Difference between the samples in the human genome project and the 100k project
In the HGP the dna sequenced was anonymous and samples were international, but this isn’t the case for the 100k project since its necessary to be able to trace back to where the dna came from
Which ethics questions are risen with the 100k project?
Who owns the data of the dna project?
Do you hand over the right o the data when sending your dna off for sequencing?
Ethical issues (in detail) from the human genome and 100k projects
Ownership of genetic information, potential discrimination, social stigmatisation, and miss use of the data (e.g - non-medical uses) When you send your DNA to the sequencing company, they hold confidential information. This could end up in the wrong hands and lead to:
-life insurance companies increasing prices if you found to have a certain gene that makes you more susceptible to a disease
-Student loans may not be given out
-Mortgage companies may not give you a mortgage
-Employers may not employ you
Identification of allele sequences enabling scientist to scan a patient’s DNA sample for mutated sequences, and also to compare the sequence of DNA bases in a patient’s gene to a normal version of the gene
Screening of embryos to detect the presence of disorders, such as cystic fibrosis Huntington’s disease, and thalassaemia could choose, which embryos are implanted, and which are destroyed
Concerns regarding the possibility of routine screening for adult onset disorder, such as Alzheimer’s disease and some cancers
Screening of embryos has led to concerns over, choosing alleles to ensure specific characteristics
Concerns that the risks of discrimination and stigmatisation could outweigh the benefits of testing
Use of genetic screening and the value of genetic counselling - sequence the genome and decide whether they have children based on this, for example, if there’s a potential of genetic disorder
Concerns regarding the storage of genetic information and its misuse - personal data could be assessed and stolen
When is dna profiling used?
To compare a dna sample from a crime scene to match with that of a suspect (forensics)
In paternity cases to determine a father
How much of the human genome is the same?
99.9% - it is the remaining 0.1% that makes an individual’s genetic profile unique
Which bits of dna does a dna profile represent?
Only non-coding portions of dna
How is a dna profile different to a dna sequence?
A dna profile only represents non-coding portions of dna
It is not the same as a dna sequence which represents all the sequences of bases in a genome.
Do we look at genes to generate a dna profile? Why?
No, they’d be more or less the same. It is only the differences in alleles that would vary, which is tiny (a matter of bases)
Is everyone’s dna profile unique?
Yes, except for identical twins
What do we aim to find in dna profiling?
Things that are different from person to person
Which parts of the genome are the parts of dna that code for proteins?
Exons
Which parts of the genome are regions of dna that don’t code for proteins?
The introns
What do introns contain?
Blocks of repeated nucleotides
Why are introns used for dna profiling?
There is a lot of variation between individuals in the introns that lie between genes because there’s more mutations
What exactly is it that causes variation in individuals, used in dna profiling?
Between exons are regions of non-coding dna called introns which contain blocks of repeated nucleotides
It is the number of times that these blocks called short tandem repeats (STRs) are repeated that produced the variation in individuals
Name for the blocks of repeated nucleotides in introns
Short tandem repeats (STRs)
Short tandem repeats (STRs)
Blocks of repeated nucleotides in introns
What are used to build up a unique fingerprint in the uk?
A number of STRs
What’s good about STRs?
They’re small and stand up well to degregation of dna over time
Give an example and explain an STR
D7S280 is an example of an STR where the base sequence “GATA” repeats on chromosome 7. Different alleles of this locus have from 6 to 15 tandem repeats of this sequence. The more times it repeats, the larger the piece of DNA will be.
Why is each individuals dna profile/genetic fingerprint unique?
Different pieces of dna move differently on the electrophoresis gel depending on their size (hyper variable dna)
What does gel electrophoresis do?
Separates dna fragments according to size
Give the stages to generating a dna profile using gel electrophoresis
- The DNA is extracted from the sample and cut into small fragments using restriction endonucleases (the bacterial enzyme that cut DNA its specific nucleotide sequences. It’s able to recognise specific sequences of DNA and cut DNA at the sequences.)
- Purify the sample.
- Fragments of DNA loaded into wells at one end of a trough containing gel.
- The gel is exposed to an electric current
- Since the fragments are negatively charged, they move towards the positive terminal.
- Smaller fragments find it easier to migrate through the pores in the gel and also travel further than largest fragments in the same time.
- The DNA becomes separated into bands according to the size of the fragments.
- Fragment size can be estimated by running a DNA ladder which contains fragments of known size alongside.
What does a dna ladder allow us to do?
Estimate the size of each fragment in a el electrophoresis
bp on a dna ladder
Base pairs
kb on a dna ladder
Kilobases
Why is it hard to compare different samples without a dna ladder?
There’s lots of variables when running a gel electrophoresis
Variables when running a gel electrophoresis that will affect the distance the dna will travel
-temperature
-strength of electrical current
-quality of gel
-concentration of dna
What is the polymerase chain reaction used for?
To increase the sample size of dna
Why is dna profiling so effective in forensics?
You only need a very small sample of dna
Why is it hard not to catch a criminal after the polymerase chain reaction?
It’s easy for a criminal to avoid leaving fingerprints at a crime scene, but it’s very difficult not to leave dna behind. However, the sample may not be large enough to use in electrophoresis, so pcr is used to magnify the sample (make it bigger)
Examples of dna left at a crime scene
Blood, rim of a glass that’s been drank out of, root of a hair
What is required to carry out numerous laboratory tests on dna?
Large samples of dna
What does pcr do?
Rapidly produced many billions of molecules from a single dna molecule
What does pcr allow to happen?
Allows tests to be carried out accurately and more rapidly regardless of the age of the sample
How can pcr be described?
Semi-conservative replication of dna in a test tube
What is the sample of dna mixed with for the polymerase chain reaction to occur?
A buffer and mixed with the enzyme dna polymerase, nucleotides, and short pieces of dna called primers
Primers used in the polymerase chain reaction
Single tranced dna typically 6-25 base pairs in length which is complimentary to the start of the sequence on the target dna
What do primers do in the polymerase chain reaction?
Act as signals to the dna polymerase to start copying
List the materials needed for dna polymerase chain reaction
-short single-stranded lengths of dna called primers
-dna nucleotides containing the four different bases (A, T, C and G)
-buffer
-heat stable DNA polymase (optimum temperature about 70 degrees) which will withstand being heated to 95 degrees without denaturing
What do we need the dna polymerase to be able to do in polymerase chain reaction?
Needs to separate the 2 strands of the double strand but the enzyme still needs to function at 95 degrees
What is the dna polymerase used in polymerase chain reaction and why?
It isn’t a normal polymerase as ours would denature at about 40 degrees and we need one that still functions at 95 degrees. The one used comes from a bacteria that lives in hot springs and actively grows at 90 degrees = taq polymerase
Stages of the polymerase chain reaction
- After placing all of the required materials in a test tube and mixing, heat to 96 degrees to separate the strands by breaking the hydrogen bonds between complementary bases. This creates single stranded DNA molecules.
- Cool to 50-60 degrees to allow the primers to attach by complementary base pairing (annealing)
- Heat to 70 degrees Celsius (optimal temperature for the enzyme) to allow the DNA polymerase to join complementary nucleotides (extension)
- Repeat 30-40 times - the new dna separates. We only have to change the temperature to repeat it - no need to add everything to the test tube again
In which direction do the primers work in the polymerase chain reaction?
5’ to 3’ (3’ to 5’ along the dna strand)
What do we set our samples up in for the polymerase chain reaction to have the temperature changing automatically?
A pcr thermocycler
What does a pcr thermocycler do?
Changes the temperature automatically
How many molecules will there be after 1, 2, 3 and 36 cycles of the polymerase chain reaction?
2
4
3
2^36 = 68 billion copies
How long would it take using the polymerase chain reaction to undergo 36 cycles?
About a day
Word to describe the increase in the number of molecules in the polymerase chain reaction
Exponential amplification
What is the polymerase chain reaction used a lot in?
Molecular biology
What happens at 96 degrees in pcr?
Separates the dna strands by breaking the hydrogen bonds
What happens at 50-60 in pcr?
Primers attach by complementary base pairing (annealing)
Word for the complementary base pairing of primers
Annealing
What happens at 70 degrees during pcr?
DNA polymerase joins complementary nucleotides (extension) - optimal temperature for this
Genetic engineering
The transfer of a gene from one organism into another, so that the gene is expressed in its new hose cell
What is the new host cell described as in genetic engineering?
Transgenic
What can genetic engineering be used for?
To introduce genes from another species into a cell
Examples of genetic engineering
Human insulin gene introduced into a bacterial cell
A bacterial gene introduced into a plant
How can a new cell make a specific new protein using genetic engineering?
We can take a gene that codes for a specific protein from one cell and transfer it to another cell. This allows the new cell to make that protein.
Good example of genetic engineering and using a gene that codes for a specific protein from one cell and transferring it to another cell
The production of insulin
What does insulin do?
It’s a hormone that controls glucose levels in the blood via a homeostatic mechanism
What do people with diabetes suffer from?
Their pancreas cells don’t produce enough insulin, therefore they have problems regulating blood glucose levels
What do people with diabetes have to do? Why?
Regular insulin injections
Their pancreas cells don’t produce enough so the glucose levels in their blood cannot be controlled
Where is insulin usually derived?
From pig pancreas
What’s the issue with obtaining insulin from a pig pancreas?
It’s from a different animal and pig insulin may not be the same as human insulin
Better way to produce insulin for people with diabetes
Genetic engineering
How is insulin generated using genetic engineering for people with diabetes?
An insulin gene is taken from a healthy human cell in the pancreas and is put in a bacterial cell. This causes the bacterial cell to produce human insulin, and when these cells divide, the gene is also replicated, so all of the cells produce insulin.
Benefit of using genetic engineering to produce insulin for people with diabetes
Lots of insulin can be produced on an industrial scale when growing a culture of bacterial cells that produce human insulin
List the basic steps in genetic engineering
- Identify and obtain the gene
- Insertion of the gene into a vector, producing recombinant DNA
- Insertion of the vector into the host cell and identification of the transgenic organism (have changed its genetics)
- Production of protein by the host cell/separation and purification of the protein
What is essentially happening when we purify a protein?
Take away the bacterial cells
Why do we need a vector in genetic engineering?
You can’t just mix the gene with bacterial cells, a vector needs to carry the gene into the cell. Normally a plasmid is used.
What is normally used as a vector in genetic engineering?
A plasmid
Why do we need to identify the exact gene needed to extract for genetic engineering?
Each cell has thousands of genes
How can a gene be identified in a cell?
Using a gene probe
What does a gene probe allow us to do?
Identify the locus of a gene
Why does a gene probe allow us to identify the locus of a gene?
Since it’s a specific segment of single-strand DNA that is complementary to a section of the gene
What occurs between the gene that we’re trying to identify for genetic engineering and the gene probe and why?
Complementary base pairing
The gene probe is a specific segment of single-strand DNA that is complementary to a section of the gene
What is also done to a gene probe? How?
It’s labelled, using a fluorescent marker for example
What can we do once we’ve located the gene needed for genetic engineering?
Can cut it out of the DNA of the protein using an enzyme
How can an identified and located gene be isolated for genetic engineering?
With either of two enzymes
a.) reverse transcriptase
b.) restriction endonuclease
Is using reverse transcriptase a direct or indirect method for isolating and getting a copy of a gene?
Indirect
Transcription
Where the cell uses the DNA strand (gene) as a template to form an mRNA molecule (complementary to the DNA strand), which is catalysed by RNA polymerase.
What is transcription catalysed by?
RNA polymerase
What does the enzyme reverse transcriptase do the opposite of?
RNA polymerase
What does reverse transcriptase do?
Copies the RNA template back to DNA
What will cells that produce insulin have in the cell that codes for insulin in the cell?
mRNA
What will cells that produce a specific polypeptide contain?
Many copies of the functional mRNA transcribed from the target gene
What are functional mRNA molecules transcribers from in terms of cells that produce a specific polypeptide?
From the target gene
Describe the process of obtaining a gene using reverse transcriptase
-cells that produce a specific polypeptide will contain many copies of the functional mRNA transcribed from the target gene
-the mRNA can be isolated and complimentary single strands of copy DNA (cDNA)
Describe the process of obtaining a gene using reverse transcriptase
- cells that produce a specific polypeptide will contain many copies of the functional mRNA transcribed from the target gene
- the mRNA can be isolated and complimentary single strands of copy DNA (cDNA) can be produced from the mRNA template using the enzyme reverse transcriptase (free DNA nucleotides used)
-DNA polymerase can then be used to make a double stranded DNA molecule. This will be an exact copy of the gene.
Which other enzyme also needs to be used when reverse transcriptase is used to obtain a gene and why?
DNA polymerase
To make a double stranded DNA molecule
What will the double stranded DNA molecule obtained by DNA polymerase be?
An exact copy of the gene
Advantages of using reverse transcriptase to obtain a gene
-this method avoids the need to locate the gene (no DNA probe needed)
-the DNA produced does not include introns cause the cDNA is copied from functional mRNA (the pre-mRNA in the nucleus that has been transcribed from the DNA has been modified (post-transcriptional processing) to produce mRNA that does not contain introns. A normal gene would contain introns. This is important since bacterial cells don’t have introns so they don’t have the enzymes to cut out the introns in their cells so it’s important that they’re already removed here
-the DNA produced does not contain any non-functional fragments
Why is it important to recognise that bacterial cells don’t have introns?
They don’t have enzymes to cut out the introns in their cells
Why don’t bacterial cells have enzymes to cut out the introns in their cells?
They don’t have introns
What are restriction endonuclease?
Bacterial enzymes (they’re not produced by eukaryotic cells) that are used to protect bacterial cells from viral DNA
What are restriction endonuclease enzymes not found in?
Eukaryotic cells
What do restriction endonuclease enzymes do?
They cut DNA (including viral DNA) at specific nucleotide sequences, and will cut DNA into many small fragments and individual genes and be isolated
In what way do restriction endonuclease enzymes cut DNA?
At specific base sequences in the DNA every time
What can we do in terms of the action of restriction endonuclease and why?
Can predict where the enzyme is going to cut the DNA
The enzymes cut the DNA at specific base sequences in the DNA every time
In which pattern does restriction endonuclease cut DNA?
Some cut straight across a DNA strand, making a blunt cut
Many make a staggered cut
What type of cut of dna by resitrction endonuclease leaves unpaired bases on both strands?
Staggered cut
Sticky ends
Unpaired bases on both strands of a dna double strand when restriction endonucleases make a staggered cut
What can we use to put DNA into another piece after restriction endonuclease has cut it?
Sticky ends
What does restriction endonuclease leave when it does a staggered cut of the double dna strand and leaves bases on both sides?
Sticky ends
Disadvantages of using restriction endonuclease
-if the recognition site occurs within the gene of interest, the gene will be broken into fragments that have no function
-eukaryotic genes contain introns, prokaryotic genes do not. If a eukaryotic gene was transferred into a bacterium it would not have the appropriate enzymes to process the pre-mRNA. The introns would not be removed after transcription and any proteins translated would therefore contain extra amino acids coded from the intron sequences. These proteins would be non-functional.
So, the process doesn’t remove introns, and bacteria wouldn’t be able to use a gene with introns to make a functional protein.
What type of cells contain introns and what type don’t?
Eukaryotic do, prokaryotic dont
What does using restriction endonuclease as an enzyme to obtain genes not remove?
Introns
Which enzyme is it more advantageous to obtain a gene with?
Reverse transcriptase
What will you want to do once you have obtained a gene using an enzyme?
Insert the gene into a vector
What is usually used as a vector in genetic engineering?
A plasmid
Explain what bacterial plasmids are
Relatively small circular DNA molecules which are found in bacterial cells and are separate from the main chromosomal DNA of the bacterium (extrachromosomal DNA)
What are the reasons that plasmids make excellent vectors for introductions engineered genes into bacterial cells?
-separate form the main chromosomal DNA
-smaller than the main chromosomal DNA
-are mobile (naturally transferred from one bacterial cell to another)
What do we mean when we say that plasmids are mobile?
They’re naturally transferred from one bacterial cell to another
What does the gene map of a plasmid show?
The location of genes in the plasmid
The restriction endonuclease recognition sites (where the restriction endonucleases will cut the plasmid)
Restriction endonuclease recognition sites
Where the restriction endonucleases will cut the plasmid
Why is it important to noice where the antibiotic resistance genes are on a gene map?
Since they’re used as markers for whether plasmids have accepted genes
What needs to be done to a plasmid in order to get the gene into the plasmid and why?
It needs to be cut since it’s circular DNA
Method for producing recombinant DNA
- Bacteria that contain the plasmid are treated to destabilise the cell walls dns breakdown the cell membrane (enzymes and detergents used)
- Plasmids are isolated from the cell debris
- The circular plasmid is cut open using the same restriction endonuclease as was used to isolate the gene. This means it has the same nucleotide sequence in its sticky ends (if the restriction endonuclease produces “blunt ends” then sticky ends can be added). The sticky ends on the gene are complementary to those on the plasmid. So, the gene can stick to the open plasmid and become inserted in there. The 2 pieces of DNA stick together. The sticky ends form hydrogen bonds between bases, which are not that strong therefore…
- DNA ligase then joins together the DNA of the plasmid and gene by catalysing the formation of phosphodiester bonds between their sugar-phosphate backbones, this permanently sticks the gene into the plasmid.
Which restriction endonuclease is used to cut plasmids?
Using the same restriction endonuclease as was used to isolate the gene
What does it mean if a plasmid is cut using the same restriction endonuclease as was used to isolate the gene?
It has the same nucleotide sequence in its sticky ends
What can be done if the restriction endonuclease produces blunt ends?
Sticky ends can be added
What type of bonds do sticky ends form?
Hydorgen
What joins the DNA of a plasmid and gene and how?
DNA ligase
By catalysing the formation of phosphodiester bonds between their sugar-phosphate backbones
What happens when DNA ligase joins the DNA of the plasmid and gene by catalysing the formation of phosphodiester bonds between their sugar-phosphate backbones?
Permanently sticks the gene into the plasmid
Plasmid that has the required gene in it
Recombinant plasmid
Recombinant plasmid
Plasmid that has the required gene in it
Do the genes always combine with the plasmid when joined together?
No - it’s hit or miss
When are sticky ends created?
When a DNA molecule is cut using a restriction enzyme
What is created when a DNA molecule is cut using a restriction enzyme?
Sticky ends
What do sticky ends consist of?
Unpaired bases on one strand of the DNA
When are the sticky ends formed complementary to one another?
When 2 pieces of DNA are cut using the same restriction enzyme
What forms when 2 pieces of DNA are cut using the same restriction enzyme?
Sticky ends that are complementary to one another
What type of cut created sticky ends?
Staggered
What can be done once the recombinant plasmid has formed?
Insertion of the vector into the host cell and identification of transgenic organism
Vector usually used in genetic engineering
Recombinant plasmids
What do we get when plasmids are mixed with bacterial cells?
Some plasmids with the gene (recombinants) and some without (plasmids close back up without the gene)
Why would some plasmids not become recombinant plasmids?
Close back up without the gene
Transformed bacteria
Have taken up the plasmids
How much of the bacteria take up the plasmid and become transformed?
As few as 1%
What 2 things must be done to obtain transgenic bacteria which contain recombinant plasmids?
- The plasmid must successfully incorporate the gene (become recombinant)
- The bacteria must successfully take up the recombinant plasmids
Recombinant plasmids
Have taken up the gene
How can successful transformation of bacteria (have taken up the plasmid) be confirmed?
-DNA sequencing
-using marker genes
What do marker genes vary with?
The type of transgenic organism produced
Example of marker genes
Antibiotic resistance genes on plasmids
What may be necessary after inserting the engineered gene into the bacterium?
To identify the bacterial cells which average successfully taken up the gene and are using it to synthesise protein
What do molecular biologists often do to identify bacterial cells which have successfully taken up he gene?
Incorporate marker genes which confer antibiotic resistance on the plasmid vector alongside the engineered gene
What do marker genes allow us to do?
Separate bacteria which have successfully then up the plasmid from those which have not
Explain how marker genes are used to separate bacteria which have successfully taken up the plasmid from those which have not
-plasmids with antibiotic-resistance genes are used, with bacteria that are not resistant as the host organism
-anti-biotic resistance genes confer resistance to one or more antibiotics such as ampicillin, tetracycline, neomycin and chloramphenicol
-the bacterial cells are cultured in a growth medium containing the antibiotic after being mixed with the plasmids, and if they have incorporated the plasmid, they also contain a gene for antibiotic resistance. They therefore break down the antibiotic and can grow. If they do not contain the plasmids, hey do it have the resistance gene and the antibiotic kills them
-surviving cells must, therefore, contain the antibiotic resistance gene
Why are antibiotic resistance genes marker genes?
Because they mark the presence of a plasmid
Types of antibiotics
Ampicillin, tetracycline, neomycin, chloramphenicol
Explain how we know that the plasmids that have been incorporated into bacterial cells are not just the original plasmids (empty plasmids) instead of being recombinant plasmids containing the gene of interest
-when we insert the gene, we ensure to use a restriction enzyme that cuts the plasmid in the middle of one of the antibiotic resistance genes so as to destroy the gene
-so, when the plasmid is cut and thee gene is inserted here, it disrupts the antibiotics-resistance gene so that it’s no longer resistant
-it its an empty plasmid, it will still be resistance, but recombinant plasmids won’t be
-of course, this contradicts the original purpose of using the marker genes to see which bacterial cells successfully took up plasmids since only the ones without the gene of interest would continue to grow in the antibiotic growth medium in this case. This is why we would use plasmids that have genes that make it resistant to more than one type of antibiotic and look for the bacteria that are resistant to one type of antibiotic but not the other. These contain the gene.
Plasmids without the gene of interest
Empty plasmids
Empty plasmids
Plasmids without the gene of interest
What would a plasmids that is resistant to only one type of antibiotic be?
A recombinant plasmid
What would a plasmid that is resistant to both types of antibiotic be?
An empty plasmid (don’t want this)
3 possible outcomes after transformation
- Bacteria that have not taken up the plasmids
- Bacteria that have taken up unaltered (non-recombinant) plasmids
- Bacteria that have taken up recombinant plasmids
Transenic
Organism that contains genetic material into which DNA rom an unrelated organism has been artificially introduced
Organism that contains genetic material into which DNA rom an unrelated organism has been artificially introduced
Transgenic
How are transgenic bacteria identified?
Using replica plating
Explain the method of replica plating
-bacteria are grown on agar in a Petri dish
-a “stamp” is placed on the first agar plate which contains one type of antibiotic (e.g - Ampicillin) and is transferred to a plate containing two types (e.g - ampicillin and neomycin) of antibiotics so the bacterial colonies can grow here
-we can compare the two plates side by side
When will the bacteria without any plasmids at all grow in the agar plates?
They won’t grow in the presence of any antibiotics
What are the bacterial colonies that grow in both types of antibiotics?
Bacteria with empty plasmids, so we don’t want these
What are the bacterial colonies that only grow in the presence of antibiotic and are what we want?
Recombinant plasmids
Do we want bacteria that grows on the plate with one type of antibiotic or two? Why?
One - these contain recombinant plasmids
What do both agar plates contain for the antibiotics test of bacteria?
The necessary nutrients as usual
What are bacterial cells with recombinant plasmids cultured in?
Large volumes in fermenters
How many transformed bacterium do we need in order to obtain large volumes of bacterial cells with recombinant plasmids?
1
Explain how the culturing of transformed bacteria is done
Bacterial cells with recombinant plasmids are cultured in large volumes in fermenters
The bacteria enzymes transcribe the inserted gene in the plasmid and translate the mRNA to produce the desired protein
Explain how insulin is made in large quantities
The bacterial enzymes transcribe the insulin gene in the plasmid and translate the mRNA they produce. Insulin is produced in large quantities and is purified for use
Concertos over genetic engineering
-plasmids are easily transferred between bacteria. There are concerns that plasmids containing antibiotic resistance genes could be passed on to other bacteria. If plasmids with antibiotic resistance genes are passed on to pathogens, this could lead to infections that cannot be treated using antibiotics. It’s possible to use different gees that aren’t antibiotic resistance genes as markers, for example ones that change colours.
-there are concerns that using fragments of human DNA could possibly transfer or activate oncogenes - when the product is contained within the human DNA, it could lead to the development of a cancer
What are genetically modified crops used for?
To increase food supply for the growing global population
What can genetically modifying crops do to them?
Can make them disease and insect resistant, enhance their nutritional value and give them drought tolerance or give a desired characteristic
What are used to produce genetically modified corps and how?
Gene technologies
By inserting a gene from one organism into another
Describe the method of genetically modifying plants using a plant cell culture
Use meristem tissue (plant stem cells) to grow new plants
1. Extract meristem and place on agar Petri dish
2. Grows into undifferentiated mass (callus)
3. Then forms little plants that can be separated and planted in soil - these will be clones (genetically identical)
Callus
Undifferentiated mass
Undifferentiated mass
Callus
What does introducing a plasmid into a callus mean?
That clones will contain the new DNA
How do we make sure that clones contain the new DNA when using a plant cell culture?
Introduce the plasmid into the callus
Give all of the ways of introducing a novel gene into plant cells
- the “gene gun” fires small spheres, often of gold or tungsten, coated with a preparation of the gene at plant cells. Some penetrate the cell wall and are taken up through the cell membrane
-electroporation
-microinjection
-using the bacterial vector Agrobacterium tumefaciens
Explain how a “gene gun” works to introduce a novel gene into a plant cell
It fires small spheres, often of gold or tungsten, coated with a preparation of the gene at plant cells. Some penetrate the cell wall and are taken up through the cell membrane.
Explain how electroporation works to introduce a novel gee into a plant cell
An electric field increases the permeability of cell membranes, enhancing gene uptake
Explain how microinjection works to introduce a novel gene into a plant cll
A membrane is pierced with an ultra-fine needle and the gene is injected into the cytoplasm or even the nucleus. This technique is much more developed for use with animal cells than plant cells.
Which technique of introducing a novel gene is more developed for use with animal cells than plant cells?
Microinjection
What is the most common method for making transgenic plant cells?
Using the bacterial vector Agrobacterium tumefaciens
What is Agrobacterium tumefaciens used as?
A bacterial vector to make transgenic plant cells
What is Agrobacterium tumefaciens?
A widespread naturally occurring soil bacteria
What does Agrobacterium tumefaciens do to plants?
Infects them
How does Agrobacterium tumefaciens infect plants?
T-DNA, a section of the bacterium’s plasmid, can integrate into the plant’s chromosomes
Plasmid genes are transcribed and translated and the auxins they produce cause a tumour, or gall, to form, giving the plant crown gall disease
Which part of the Agrobacterium tumefaciens bacterium’s plasmid can integrate into the plant’s chrosomes?
T-DNA
What disease can plants get from the Agrobacterium tumefaciens bacterium?
Gall disease
What’s the useful thing that can be done with Agrobacterium tumefaciens?
It has the ability to introduce new genetic material into the plant cell
What is the genetic material that is introduced into a plant by Agrobacterium tumefaciens called?
T DNA (transferred DNA)
T DNA (transferred DNA)
The genetic material that is introduced to a plant cell by Agrobacterium tumefaciens
Where is T DNA located?
On a Ti plasmid (tumour - inducing plasmid)
What is located on a Ti plasmid?
T DNA
What is the Ti plasmid used for when introducing new genetic material into a plant cell?
As a vector
What can be done to chosen genes on a Ti plasmid?
Chosen genes can be spliced into the plasmid
How can genes be transferred into plant cells using Ti plasmids?
By splicing chosen genes into the plasmid
What’s done after genes are transferred into plant cells using Ti plasmids?
The plant cells are subsequently grown in tissue culture and regenerated into plants which express these introduced genes
Explain the stages in how Agrobacterium tumefaciens is used to introduce a novel gene into a plant cell
- Ti plasmid is extracted from the Agrobacterium tumefaciens
- Restriction enzyme is used to cut the plasmid and remove the tumour-forming gene
- A section of DNA containing a gene for disease resistance is located and isolated using the same restriction endonuclease and incubated within the restriction endonuclease
- The gene is inserted into the plasmid to form a recombinant Ti plasmid, replacing the tumour-forming gene. DNA ligase is used to join the donor and vector DNA together. Use sticky ends to put the DNA into the plasmid.
- The bacterial cell is introduced into plant cell. The bacterial cell divides and gene is inserted into plant chromosome. The inserted T DNA carried the new gene.
= new DNA is part of the plant’s genome
=cuttings from the plant will contain the new gene - Transgenic plant cells are grown in tissue culture and transformed plants are regenerated. The result is a plant with the new trait
What is used to cut a Ti plasmid and remove the tumour-forming gene?
Restriction enzyme
What joins the donor and vector DNA together in a Ti plasmid?
DNA ligase
What do we use to put DNA into a plasmid?
Sticky ends
Give 2 examples of crops that are frequently genetically modified
Soya beans
Tomatoes
How have soya beans been genetically modified?
Made to be herbicide-resistant so that the crops can be sprayed to remove weeds without inhibiting their growth
What are the 2 examples of genetically modified tomatoes?
BT tomatoes
Antisense tomatoes
Explain how and why BT tomatoes have been genetically modified
Bacillus thuringiensis is a bacterium that lives in soil and contains a plasmid with a gene that codes for a protein that acts as an insecticide. The insecticidal proteins are made in the leaves that insect’s eat. There’s therefore no need to spray crops with insecticides, which protects all the unintended targets of applied insecticide
Explain how and why Antisense tomatoes have been genetically modified
Tomatoes ripen naturally when they produce the enzyme polyglacturonase, which breaks down the pectin in their cell walls. But, if they are transported long distances from their supplier, they may over-ripen and not be suitable to sell. To overcome this, Agrobacterium tumefaciens was used to introduce a second copy of the polygalacturonase gene into the tomato plant, but this copy has a base sequence complementary to that of the normal gene (i.e - it was an “antisense” gene). The mRNA transcribed from the antisense gene is complementary to the mRNA strand of the original gene. This two types of RNA base pair in the cytoplasm to form a double-stranded molecule. This prevents the mRNA of the OG gene from being translated and blocks the production of the enzyme.
What have questions been raised about since the introduction of genetically modified food plants?
Potential risks, labelling, nutritional properties and effects on the environment
Benefits of genetically modified crops
Higher crop yields
A substantial reduction in pesticide use
Improved food
Better keeping qualities
“Pharming”
Explain how higher crop yields are obtained as a result of genetically modifying crops
Ever more crops are lost to disease and as the world’s climate changes, to droughts and floods. Incorporating genes for insect, fungus and worm resistance or for drought or salt tolerance are likely to increase crop yield. Introducing genes that confer resistance to herbicide is likely to decrease plant loss in the field
Explain why there will be a substantial reduction in pesticide use due to genetically modified crops
Genes for fungal pathogen resistance and resistance to insect attack have the additional advantage of reducing the quantities of pesticides applied to farmland
Explain and give an example of how food can be improved with genetic modification
Nutritional quality can be enhanced, as with Golden Rice, which contains an added gene to increase the content of vitamin A precursors and prevent blindness in children in some parts of the world. Foods can also be enhanced with improved flavour and better keeping qualities = less waste
What does better keeping quality of plants that have been genetically modified lead to?
Less waste
Explain “pharming”
Refers to the production of pharmaceutical molecules in genetically modified crop plants. Plants have been modified to make antibodies, blood products, hormones, recombinant enzymes and human and veterinary vaccines.
What’s the issue with the cultivation of maize, rice and wheat?
It requires huge quantities of nitrogenous fertilisers
What has the cultivation of cereals done?
Disrupted the global nitrogen cycle and has had severe environmental effects.
What is being done in terms of genetic engineering to combat the disruption of the global nitrogen cycle in the cultivation of cereals?
Attempts continue to try to make transgenic cereals that contain the nif (nitrogen fixing) genes from nitrogen-fixing bacteria.
Why do we want plants with nitrogen fixing genes?
The plants would then make their own fertiliser and less would be added artificially. This would make a value contribution to restoring damaged habitats
Concerns about genetically modified crops
Gene transfer
Pest resistance
Marker genes
Biodiversity decrease
New proteins
“Organic farming”
Economic concerns
Explain the problem with gene transfer after genetically modifying crops
Pollen from GM plans might transfer genes to wild relatives. In this way, it is feared that herbicide resistance might spread to wild plants and produce “superweeds”, which can’t be killed if they grow out of control. If GM crops do not have wild relatives in the UK (e.g - potatoes), this fear may be unfounded
Explain the concern with genetically modified crops having insect resistance
Plants with introduced genes that enable them to resist insect attack may lead to a population of resistant insect or fungal pests. Long-term field trials will establish whether these concerns are well-founded. However, if crops are modified to synthesise more than one pesticide, is it likely that resistance to 2 would develop simultaneously
Explain the concerns relating to marker genes for genetically modified crops
Genetically modified organisms contain marker genes, some of which confer antibiotic resistance. There is concern that these genes may be transferred to the bacteria in the intestine of the consumer.
Explain how genetically modified crops could decrease biodiversity
Plant breeding may fall into the hands of a few commercial companies, limiting the number of crop varieties available to the farmer. This could lead to the elimination of old varieties. Reduction in biodiversity decreases the range of potentially useful genes. Same with monoculture crops - 1 species growing over wide areas.
Explain the concerns relating to new proteins in genetically modified crops
It is claimed that there may be adverse health effects from eating a crop that is expressing a new gene, making a new protein. No evidence in any organism has supported this claim.
Explain the concerns in terms of organic farming with genetically modified crops
It is claimed that pollen from genetically modified crops could compromise organic crops
Explain the economic concerns about genetically modified crops
Genetically modified organisms are subject to intellectual property law and it is featured that the associated expense will be borne by the farmer
Are there any GM crops grown in the UK?
No - although some are imported
The methods of what does tissue engineering se?
Biochemistry, cell biology, engineering and materials science
What does tissue engineering use the methods of biochemistry, cell biology, engineering and materials science to do and how?
To repair, improve or replace biological functions by the replacement of tissues or organs
Goal of tissue engineering
To produce “off the shelf” bio-artificial organs and to regenerate injured tissue in the body
What has tissue engineering allowed?
The replacement of many tissues and organs
What is tissue engineering hoped to treat in the future?
Diabetes
Traumatic spinal injury
Duchenne muscular dystrophy
Heart disease
Vision and hearing loss
What does tissue engineering involve?
Inducing living cells to grow on a framework of synthetic material to produce a tissue such as skin
Applications of tissue engineering examples
Produce a tissue such as skin
Blood vessel replacement
Bone and cartilage repair
Treatment of degenerative nerve disease
In theory, can all cells exist independently of the body? Explain
Yes, provided they are supplied with the nutrients they require
What do most cells do?
Differentiate into cells which have specific functions such as nerve or muscle cells
What happens to most specialised cells once they’ve differentiated into a specialised cell?
They do not divide again
Telomeres
The repeated nucleotide sequences on the ends of chromosomes
What happens to telomeres at each cell division?
They shorten, and limit cell morality
How have the life of differentiated cells been extended?
By developing techniques for elongating telomeres
How can cells be separated from tissues? Give examples
With enzymes
E.g - trypsin and collagenase
When can cells be used to grow tissue replacements?
Following the elongation of their telomeres
What can be done with cells following the elongation of their telomeres?
They can be used to grow tissue replacements
What are the preferred materials for tissue engineering?
Stem cells
Where can stem cells be extracted from?
Blood and from solid tissues
How are cells for tissue engineering classified?
By their source
Autologous cells
From the same individual
Advantage and disadvantage of using autologous cells for tissue engineering
Advantage = fewest problems with rejection and pathogen transmission
Disadvantage = not always available (e.g - patient has a genetic disease or severe burns or is very ill or old)
Allogeneic cells
Come from the donor of the same species
Xenogeneic cells
Come from another species
What do we need to be careful with with xenogeneic cells? Explain
They’ve come from another species so we need to be careful of viral sequences. They may be harmless on one species but dangerous in humans.
Synergeneic or isogeneic cells
Form genetically identical organisms
What is done to cells on a scaffold during tissue engineering?
They’re “seeded” on to the scaffold
Scaffold
An artificial structure that can support a 3D tissue
An artificial structure that can support a 3D tissue
Scaffold
What features must scaffolds used in tissue engineering have?
Must allow cells to attach and move
Must deliver and retain cells and biological molecules
Must be porous to allow diffusion of nutrients and waste products
Must be biodegradable and be absorbed by the surrounding tissues. The rate it degrades should match the rate of tissue formation to eventually it will break down leaving the “neotissue”
What should the rate that a scaffold degrades be the same as when used in tissue engineering?
Should match the rate of tissue formation, so eventually it will break down leaving the neotissue
What is the technique of growing cells in a laboratory called?
Tissue culture
Tissue culture
The technique of growing cells in a laboratory
What do cells that are grown in tissue culture do?
Form cell lines that are clones, as all the cells derived from a single parent cell are genetically identical.
Why are all of the cell lines formed from cells in a tissue culture clones?
As they’re all derived from a single parent cell and are therefore genetically identical
What are cell lines generated in a tissue culture used to do?
Produce cloned tissue sample and, in some cases, to generate organs
What is the production of cloned material referred to as?
Therapeutic cloning
Why is the production of cloned material referred to as therapeutic cloning?
To distinguish it from cloning whole organisms (i.e - reproductive cloning)
Major advantage of therapeutic cloning in a tissue culture
A patient is unlikely to reject tissue or organs cloned from their own cells
What must cells be given during the period of tissue culture?
Oxygen, nutrients, growth factors and the correct pH, humidity, temperature and water potential
Which method is normally adequate in tissue culture and what may be needed as structures get larger?
Diffusion
Capillary networks needed as structures get larger
What have cell cultures been used for some time for?
Medical and research purposes
Examples of medical and research purposes of cell cultures
In the culture of viruses for vaccine production and also in the production of monoclonal antibodies
What are sometimes needed in tissue engineering?
Special physical or chemical stimuli
Examples of specialised physical or chemical stimuli sometimes needed in tissue engineering for the correct structures to differentiate
Chondrocytes need low O2 concentration, mimicking their development in skeletal tissue
Endothelial cells need shear stress to mimic blood flow in blood vessels
Cardiovascular tissue such as heart valves need mechanical stimuli such as pressure pulses to simulate their development
Why are special physical or chemical stimuli sometimes needed in tissue engineering?
For the correct structures to differentiate
Stem cells
Unspecialised cells that can develop into many different cell types
Unspecialised cells that can develop into many different cell types
Stem cells
What happens when a stem cell divides by mitosis?
Each daughter cell can either remain a stem cell or become another type of cell with a more specialised function
Examples of specialised functions stem cells can develop after mitosis
Muscle fibre
Red blood cells
Liver cell
Nerve cell
Types of stem cell
Totipotent cells
Adult tissues containing stem cells
Induced pluripotent stem cells
Embryonic stem cells (ESC)
What can totipotent cells do?
Can form every cell type in an organism
Example of a totipotent cell
Zygote
How do we know zygotes are totipotent cells?
They form all cell types in the body and the cells that support embryonic development (e.g - placenta)
What are induced pluripotent stem cells?
Adult cells reprogrammed to become these
iPSCs
Induced pluripotent stem cells
Where are embryonic stem cells found?
In 3–5 day old embryos
What can embryonic stem cells be described as and why?
Pluripotent
Can form every cell type in the body
Examples of adult tissues containing stem cells
Bone marrow, muscle and brain
where exactly in bone marrow, muscle and brain are there adult tissues containing stem cells?
In the “stem cell niche”
What can stem cells obtained from adult tissues be used for?
Replacing cells lost through normal wear and tear, injury or disease
How can stem cells from adult tissues be described?
Adult/multipotent stem cells
Multipotent stem cells
Adult stem cells that cannot form all cell types
What type of stem cells cannot form all cell types?
Adult/multipotent stem cells
List the possible potentials of stem cells
For tissue engineering
For cell-based therapies
To screen new drugs
To develop model systems
To investigate the events that occur during human developments
How can stem cells be used in tissue engineering?
To regenerate tissues and organs
Give an example of using stem cells for tissue engineering
Regenerating bone using cells derived from bone marrow stem cells
Explain how stem cells can be used for cell based therapies to treat disease
The need for transplantable tissues and organs far outweighs the available supply. Stem cells stimulated to differentiate into specific cell types are a renewable source of replacement cells and tissues to treat diseases.
How have long been used to screen potential anti-tumour drugs?
Cancer cell lines
Why would using stem cells be better for screening new drugs than using cancer cell Ines to screen potential anti-tumour drugs?
Stem cells allow drug testing in many more different cell types
Why would we use stem cells to develop model systems?
To study normal growth and identify the causes of birth defects
Explain how stem cells could be used to investigate the events that occur during human development
Investigate how gene switches turn differentiated stem cells into differentiated cells and form tissues and organs
What do gene switches do to stem cells?
Turn differentiated stem cells into differentiated cells and form tissues and organs
What’s the main advantage of using stem cells?
They will make the acute problem of shortage of organs for transplant less significant
Give the advantages of using embryonic stem cells over adult stem cells
(ESC first, adult stem cells second)
Can become any cell type, can become a specific cell type or types
Isolated in useful numbers (e.g: about 100 ESCs in a blastocyst), fewer so isolation more challenging
Easily grow large numbers, culture techniques less developed
Advantage of using adult stem cells over embryonic stem cells
For adult stem cells, the tissues are derived from own adult stem cells so they’re less likely to provoke immune responses.
Patients receiving ESC-derived tissue need life-long immunosuppressants with possible side-effects
Disadvantages of using stem cells
Techniques for extracting, cultures and manipulating stem cells are still under development and the behaviour of cells cultures is not always predictable. As a result, currently, products of stem cell technology, such as the artificial trachea, are expensive are rare
The use of stem cells is very new and so long-term studies have not yet been possible. There are concerns related to the premature aging of cells are other as yet unpredictable events
Explain all of the issues surrounding the use of embryonic stem cells
-recent registration allows researchers to create embryos for research, but prior to this, they used “spare embryos” from in vitro fertilisation. Some people argued that creating embryos specifically for research contravenes the principle that human life should never be created as a means to an end. These embryos, though, cannot legally be transferred to a uterus so a new individual could never be born from one of them
-the moral status of the embryo (the Catholic Church believe life begins at conception and other religious traditions believe that as a foetus develops for example as it acquires a nervous system, its rights increase)
-some people think its never justified as we can use adult stem cells and iPSs instead
-fear of stem cells leading to human clones
Why is it still believed that embryonic stem cells are important despite the controversies?
-they will clarify fundamental biological mechanisms
-they will indicate which types of stem cell will be most useful in cell-based treatments
-a pre-14-day embryo is a ball of cells with no possibility of independent existence, so its use it justified
How can gene therapy be used?
To treat genetic disorders
How can gene therapy be used to treat genetic disorders?
By inserting functional DNA sequences into cells to counteract the effect of a defective gene
When do genetic disorders occur?
When the gene isn’t working properly
What is the thinking behind gene therapy?
Genetic disorders occur when the gene isn’t working properly - a functioning one replacing it may fix it
How can genetic diseases can be treated?
By replacing genes
Or
Replicating the function of genes using drugs
2 possible methods of replacing defective genes
Somatic cell therapy
Germ line therapy
What will somatic cell therapy not do?
Prevent the condition being passed on
What type of method for replacing defective genes using gene therapy is rarely used?
Germ line therapy
Aim of gene therapy
To treat a genetic disease by replacing defective alleles in a patient with copies of a new DNA sequence (cloned from a healthy individual)
Where does the new DNA sequence that replaces the defective alleles during gene therapy come from?
Cloned from a healthy individual
What does gene therapy use to introduce the DNA into the target cells?
A virus as a vector or
A plasmid as a vector or
Injection of naked plasmid DNA
2 main approaches of gene therapy
Somatic cell therapy
Germ line therapy
What can somatic cell therapy be described as?
Therapeutic
In what type of cell therapy are genetic changes not inherited?
Somatic cell therapy
How come genetic changes are not inherited in somatic cell therapy?
They’re not inherited in the daughter cells of the treated cells, and do not appear in future generations
What type of cell therapy has to be repeated regularly?
Somatic cell therapy
Why will somatic cell therapy have to be repeated regularly?
As the treated cells become worn out and are replaced by the body with no cells that do not contain a working copy of the gene
What are therapeutic genes transferred into in somatic cell therapy?
Into the somatic (body) cells in the organ/tissues affected
How is DNA introduced into target cells during somatic cell therapy?
By a vector
Where does germ line therapy introduce the correct genes into?
Germ-line cells
Germ line cells examples
Oocyte (eggs), sperm
Why does germ line therapy introduce the corrective genes into germ line cells such as the oocyte?
So that the genetic condition is inherited
How are sperm/eggs modified during germ-line therapy?
By introducing functional genes
What happens once corrective genes have been introduced into the germ line during germ line hearpu?
Genes are integrated into the genome
What happens once genes have been integrated into the genome in germ line therapy?
Will be passed on to future generations
Why is germ-line therapy controversial?
Genes interact with each other. Some are switches which control other genes. Changing one gene or set of genes in the oocyte has the potential to cause unpredictable effects in future generations.
Also, in many cases, germ-line therapy can be avoided through IVF-screening to see whether the defective gene is there and only used embryos that don’t have the genetic condition
Give an example of a use of gene therapy
Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD)
A recessive sec linked form of muscular dystrophy affecting up to 1 in 3500 live male births
What is DMD causes by?
One or more mutations in the dystrophin gene
How many exons which code for proteins does the dystrophin gene have?
79
What does a mutation in any of the 79 exons of the dystophin gene cause?
It alters how the gene is transcribed as mRNA
Explain exactly what happens when a mutation occurs to the dystrophin gee
A mutation alters how the gene is transcribed as mRNA. This results in the failure to produce dystrophin
Why do we need dystrophin?
It’s an important structural component of muscle tissue
Results of not producing dystorphin with DMD
Severe wasting of the muscles and sufferers are often wheelchair bound by the time they reach teenage years (life expectancy = 27 years)
Where does the gene for dystrophin production sit?
On the x-chromosome
Explain when dystrophin will be made in terms of its position on the X chromosome
If a normal gene for dystophin is present, then the protein will be made
If the gene is missing or altered then dystophin may not be produced t all or only in abnormal form, resulting in Duchenne muscular dystrophy
What type of gene is dystorphin?
Recessive
Why does DMD mostly affect males?
Since females have a “spare” X chromosome
What are 1/3 cases of DMD?
Random mutations that are not inherited
Which drug is in development to treat DMD?
Drisapersen
How does the drug drisapersen aid to treat DMD?
By introducing a “molecular patch” over the exon(s) with the mutation making the gene readable again
What forms with drisapersen?
A shorter form of dystrophin, but one thought to be more functional than the untreated version
What is the treatment of DMD with drisapersen known as?
Exon skipping
What happens to the reading frame of the transcript with drisapersen?
It’s restored
Describe the dystrophin formed for someone with DMD vs when using exon skipping to reframe transcripts using drisapersen
DMD = dystrophin translation stops prematurely
Exon skipping = internally deleted, but partially functional dystophin
Describe the mode of action of drisapersen
Drisapersen is a 50-nucleotide sequence (antisense oligonucleotide) sequence that is complementary to the mutated sequence
It binds to the mRNA over the exon with the deletion
That portion of RNA therefore becomes double stranded and is removed as the mature mRNA is formed, since the ribosome is unable to translate that potion of the mRNA and therefore skips the mutation
This produced a shorter, partially functional dystrophin protein molecule
How is drisapersen delivered to people with DMD?
It’s delivered in subcutaneous injections
Why is drisapersen not used all of the time to treat DMD?
Clinical trials have not yet provided clear evidence as to the best age for treatment or for its length
What are other approaches to DMD treatment except for using the drisapersen drug?
Gene therapy - a shortened version of the healthy gene has been designed because the normal gene it too big to put into a virus
Research into stem cell treatment
Explain how somatic cell therapy is done using a virus
- Cells are removed from patient
- In the laboratory, a virus is altered so that it cannot reproduce
- A gene is inserted into the virus
- The altered virus is mixed with cells from the patient
- THe cells from the patient become genetically altered
- The altered cells are injected into the patient
- The genetically altered cells produce the desired protein or hormone
Where is it hard to get genes into?
Affected cells
Explain the somatic gene therapy used to treat DMD
- The modified dystophin gene is inserted into an AAV vector
- The viral vector inserts the dystopian gene into the nucleus of the muscle cells
- The muscle cell cell begins to produce dystophin protein
Describe tissue engineering
Complex and difficult
Why do we need host cells when using a culture of viruses in tissue engineering?
Viruses replicate by taking over a host cell
How to viruses replicate?
By taking over a host cell
What type of cells do we need when we create a culture of viruses?
Host cells
What do immune systems often do to organ transplants and why?
Reject them
The tissues don’t match so they’re seen as foreign
What does growing an organ via tissue engineering avoid? Explain
Immune systems can reject organ transplants because tissues don’t match and are seen as foreign. Growing an organ avoids his because the tissues will match.
What type of cells are turned into what for induced pluripotent stem cell?
Differentiated cells turned into stem cells
What can induced pluripotent stem cells then do?
Can grow into all types of cell
Why is there a problem in getting cells to grow and how is this avoided?
Specialised cels don’t divide once mature
Stem cells avoid this problem - they have ht potential to differentiate into any type of cell
What are stem cells currently being used to treat?
Leukemia
What is done about sticky ends when using reverse transcriptase to isolate a gene?
They can be added at the end to each DNA strand
How does biodiversity decrease when introducing insect repellent genes?
Food chains impacted by killing insects
How would GM plants transfer genes to wild relatives?
Via pollen
Aims of the human genome project
Identify the position of genes in the human genome
Improve understanding, diagnosis and treatment of genetic disorders
What does a DNA ladder contain?
Fragments of known lengths alongside
When can’t we be sure of a result on a gel electrophoresis with a DNA ladder?
When we have blurred lines
What kind of sequencing was used in the 100k project?
Next generation sequencing (NGS)
Things possible after 100k project
Prenatal diagnosis
Understanding disease
Gene therapy
What’s the main ethical dilemma with the human genome project and 100k genome project?
Rights to access of genetic information
