gene technology

what is the genome

all the genes / genetic material of an organism

what is the proteome

the full range of proteins that can be produced by an organism at a given time

what is larger, the genome or the proteome

the proteome

what are the benefits of developing a complete map of the human genome

• develop a better understanding of which genes are responsible for certain genes

• production of more effective medicines for genetic diseases and cancer

• aids the development of vaccines

• tracing evolutionary relationships

• allows for genetic screening

suggest why it might be difficult to work out a mammals proteome from its genome

proteins are only produced when the genes for them are switched on

much of the genome consists of introns (non-coding DNA)

production of some proteins is influenced by multiple genes

scientists can gain information about antigens from sequencing the proteome of pathogens, why might this be useful

antigens are found on the surface of pathogens and pathogens can cause disease

knowledge about antigens may make it easier to identify pathogens

knowledge about the antigens found on pathogens may facilitate the production of effective vaccinations and medical treatments for particular diseases

suggest and explain how identifying the parasite that causes malarias’s genome can be used to speed up the development of a vaccine

the genes showing the largest level of variance are likely to be the antigens (because antigens vary to try and evade the host’s immune system)

the identification of these vary genes, allows scientists to specifically focus on this area of the genome, instead of wasting their time looking at areas of the genome that don’t affect antigen production

once the antigenic genes have been worked out, scientists can then synthesise the proteins produced by these genes and inject them into people (as vaccines) to determine whether it causes an immune response

why is it hard to determine the human proteome (eukaryotes)

eukaryotes have introns in the DNA (non coding sections of DNA)

eukaryotes have regulatory DNA where genes can be switched on by transcription factors to produce proteins- expression (on/off) of these genes is constantly changing)

why is the proteome of a prokaryotic pathogen easier to determine than the proteome of a human

the prokaryotic proteome is smaller than the human proteome

prokaryotes don’t have introns (non coding DNA), whereas eukaryotes have many introns incorporated into it

humans have more regulatory DNA than prokaryotes (DNA where genes can be switched on by transcription factors to produce proteins- expression (on/off) of these genes is constantly changing)

why is it necessary to sequence the DNA of several organisms of the same species to develop an accurate genetic template of the species

individuals show genetic variation, so may have differences in there genes and alleles

there may be mutations within the DNA sequence of an individual

using a larger sample size/more individuals provides a more accurate representation of the population

how can DNA sequencing be used to determine evolutionary relationships

species that are more closely related will have more similar DNA/RNA sequences and protein sequences

DNA for human genome sequencing projects uses blood sample, which cells are used and why

white blood cells

these are the only cells in the blood that have nuclei (platelets and RBC’s don’t)

what are the benefits of automated DNA sequencing methods compared to the original manual methods

algorithms are used to analyse data on genetic sequences- reduces the risk of errors in the sequencing process

scientists can make comparisons of gene sequences with known sequences stored in the data base

can make genome comparisons faster

computers can immediately identify new genes and alleles

what are the advantages of making scientific data public, prior to publication

any medical treatments developed by using the public data would be cheaper (don’t have to pay to use the data)

speed of the research process would be faster because other scientists can access the information for collaboration

what are the disadvantages of making scientific data public, prior to publification

have to rely on public funding- charity or government funding

the data could be used by other researchers for unethical and illegal research

what does DNA sequencing allow for

the base sequence of an organism to be identified and recorded

the genetic code is ‘universal’, what does this mean

almost every organism uses the same 4 nitrogenous bases (ATCG)

the same codons code for the same amino acids in all living things (genetic information is transferrable between species)

what is recombinant DNA (rDNA)

DNA that has been artificially changed by combining lengths of nucleotides from different sources (different species e.g. bacteria)

this process works because the genetic code is universal (same 4 bases and codons will code for the same amino acids)

Why are bacteria able to use human DNA to produce human proteins

• The genetic code is universal (same for prokaryotes and eukaryotes)

• The mechanism of transcription and translation are universal

what happens in genetic modification

fragments of DNA from one organism have to be transferred into another organism

this results in a genetically modified organism which will contain recombinant DNA (rDNA)

what are some applications of recombinant DNA technology

Production of recombinant proteins (such as insulin) from bacterial cells

Gene therapy- inserting healthy copies of mutated genes into human cells

Pest-resistant crops- better yield

Herbicide-resistant crops- better yield

More nutritious crops (with added nutrients)

increased growth hormone production in farmed animals- better yield

describe the process of in vivo gene cloning

The desired gene/DNA fragment is identified

The gene/DNA fragment is isolated and extracted (isolation)

The gene/DNA fragment is then inserted into a plasmid vector (insertion)

allow the bacteria to multiply (transformation)

The bacteria cells that have taken up the gene/DNA fragments are identified using genetic markers (identification)

The genetically modified cells are then cloned (growth/cloning)

describe the process of in vitro cloning

the desired gene / DNA fragment is identified

the gene / DNA fragment is isolated and extracted (isolation)

DNA fragments are amplified in the polymerase chain reaction (PCR)

how is the desired gene/DNA fragment isolated and extracted

the gene with the specific characteristic that is required can be obtained in three ways:

• extracting a gene from the DNA of a donor using restriction endonuclease enzymes

• using reverse transcriptase to synthesise a single strand of complimentary DNA from the mRNA of a donor organism

• synthesising the gene artificially using the gene machine

what are restriction endonucleases

they are enzymes found in bacteria used as a defence mechanism against viruses- where they cut the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule

the restriction endonucleases are named according to the bacteria they are sourced from

how can a gene be extracted from the DNA of a donor using restriction endonucleases

the restriction endonuclease enzyme will cut a DNA double strand at the same place at a particular recognition sequence- normally around 6 base pairs

this can form sticky ends where the sugar-phosphate backbone is cut in an uneven/staggered way

or it can form blunt ends where the sugar-phosphate backbone is cut straight across

why are sticky ends preferred over blunt ends

when the DNA is cut to produce “sticky ends", two complimentary ends are created

This means that only DNA sticky ends produced with the same restriction endonuclease can join together

With blunt ends, there is no specificity to the cut

this means that any strand can join with any other strand are the ends are not specific and complementary

how can reverse transcriptase (enzyme) be used to synthesise a single strand of complimentary DNA from the mRNA of a donor organism

Use the mRNA that was transcribed for the desired gene

Once isolated, the mRNA is combined with a reverse transcriptase enzyme and nucleotides to create a single strand of complementary DNA

add DNA primer that will bind to the mRNA strand which signal reverse transcriptase where to start synthesising the DNA

this forms a DNA strand complimentary to the RNA strand

where can reverse transcriptase enzymes be sourced from

retroviruses

why is isolating the desired gene using reverse transcriptase preferred over using restriction endonucleases

mRNA doesn’t contain introns (non-coding DNA)- introns are spliced

how can e gene with the specific characteristic that is required can be obtained via artificial synthesis using a “gene machine”

due to technological advancements, scientists are becoming more aware of the proteome (base sequences for our proteins)

this makes it possible to synthesise genes artificially

as we know which amino acids are required, scientists can use computers to generate the nucleotide base sequences for genes to produce the proteins that we want

short fragments of DNA are produced first which are joined to make longer sequences of nucleotides, which are then inserted into vectors (such as plasmids)

what happens after the gene/DNA fragment is isolated

promoter and terminator regions are added to the fragments of DNA to ensure replication

what two ways can DNA fragments be amplified

in vivo cloning- use of plasmids and bacterial cells

in vitro cloning- PCR

how are the DNA fragments inserted into plasmids (insertion)

restriction endonucleases are used to cut plasmids and produce sticky ends that are complimentary to the desired gene’s sticky ends

the isolated DNA fragment with the correct sticky ends, promoter region and terminator region are mixed together with DNA ligase

DNA ligase catalyses the condensation reaction where phosphodiester bonds are formed between the nucleotides on two separate DNA strands

some plasmids will take up the gene and form recombinant plasmid DNA

what happens in transformation in in-vivo cloning

the vector (recombinant plasmid DNA) is transferred into bacterial host cells

to do this the cell membrane of the host cell must be more permeable- which can be done by mixing the bacterial cells with calcium ions and a sudden increase in temperature

only a few bacterial cells actually take up the recombinant plasmid DNA

these bacterial cells can be identified by using markers

why are bacterial cells used in in-vivo cloning

bacterial cells are used because the can divide and reproduce rapidly via binary fission and they are relatively easy to culture

why do we need to identify transferred bacteria

not all of the host cells (bacteria) will take up the recombinant plasmid DNA

this can be due to:

• the recombinant plasmid DNA doesn’t get inside the cell

• the plasmid rejoined to itself before the DNA fragment could join

• the DNA fragment sticks to itself, rather than to the plasmid

how are bacterial cells that have taken up the recombinant plasmid DNA identified (identification)

there are different methods:

• replica plating

• fluorescent markers

• enzyme makers

how can replica plating be used to see if plasmids have taken up the DNA fragment

plasmids have genes for ampicillin and tetracycline antibiotic resistance

depending on which restriction endonuclease is used to cut stick ends, the gene for either ampicillin or tetracycline antibiotic resistance could be removed

BamHI restriction endonuclease removes tetracycline resistance but ampicillin resistance remains

method:

individual bacteria cells are plated onto the master plate, replicating to form colonies

a replica plate is cultured with ampicillin, leaving only cells that contain plasmids with ampicilin resistance

a second replica plate is cultured with tetracycline, leaving only cells with ampicillin and tetracyclie resistance, so these plasmids haven’t taken up the DNA fragment

the cells with ampicillin resistance, but no tetracycline resistance are the desired cells

what are some disadvantages of replica plating

• creating colonies of bacteria → antibiotic resistance

• slow process

• wasteful process

how can fluorescent markers be used to identify plasmids that have taken up the DNA fragment

plasmids containing a green fluorescent protein (GFP) incorporated from a jelly fish can be used

a restriction endonuclease cleaves a site at the GFP region should be used

plasmids that take up the DNA fragment / gene will not fluoresce under UV light → they are the bacterial cells (called transformed bacteria) that will be cultured, so the desired gene is cloned

plasmids that fluoresce under UV light will be destroyed

how can enzyme markers be used to identify whether a plasmid has taken up the DNA fragment

another gene marker is the gene that produces the lactase enzyme

lactase breaks down lactose in X-Gal and one of those products is blue in colour

a restriction endonuclease that cleaves the lactase producing gene should be used

if the insertion is successful and transformed bacteria is produced, then when the bacterial cells are cultured with X-Gal, the substrate will not turn blue as lactase isn’t produced → these are the bacterial cells that will be cultured so the desired gene will be cloned

what are transferred bacterial cells

bacterial cells that have taken up the recombinant plasmid DNA

what happens in the growth/cloning stage of in vivo cloning

the transformed bacteria will have been identified

these are the bacteria that:

• aren’t fluorescent under UV

• when cultured with X-Gal, the substrate won’t be blue

• have only either ampicillin or tetracycline resistance (not both)

these bacterial cells will then be cultured in a fermenter, every time the bacterial cell divides via binary fission then the desired gene will be cloned

these bacterial cells with then synthesise the protein coded for by the desired gene

what is the purpose of PCR

to produce large quantities of specific DNA fragments from small quantities of DNA

what piece of equipment is used in PCR to change the temperature and control the amount of time sent at each stage

thermocycler

which is an automated machine

what are primers

short sequence of single stranded DNA

they are complimentary to the start and end of the DNA being copied

they provide a starting point for DNA polymerase

what does each PCR reaction require

target DNA

primers

DNA polymerase- Taq DNA polymerase (attained from bacteria that live in hot springs because this enzyme has a much higher optimum temperature so it won’t denature at high temperatures (72.) in the thermocycler)

Free DNA nucleotides- construction of the DNA strands

buffer solution- provide the optimum pH for the reactions to occur in

thermocycler

describe the principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments.

denaturation (heat to 95°C)

annealing (cool to 50-65°C)

extension (heat to 72°C)

what happens in the denaturation stage of PCR

DNA sample is heated to 95°C which causes hydrogen bonds between complimentary base pairs to break

this results in the DNA separating into two single strands

what happens in the annealing stage of PCR

the temperature is lowered to 50-65°C, allowing the primer to bind (anneal) to the 3’ end of the target DNA

the temperature is cool enough for hydrogen bonds to reform so that the primer is held in place

primers prevent the two DNA strands from rejoining/re-annealing

the primer provides a starting point for DNA polymerase, as it can only add nucleotides to the existing strand

what happens in the extension stage of PCR

the temperature is increased to 72°C, which is the optimum temperature for Taq Polymerase

Taq polymerase adds free DNA nucleotides (A,T,C,G) and forms phosphodiester bonds between adjacent nucleotides → forms a new DNA polymer

why is it better to start PCR with mRNA rather than DNA

mRNA is shorter than DNA and more specific to the genes you want to amplify

introns in DNA are spliced/removed in mRNA

what are the advantages of PCR/ in vitro cloning

automated machine (thermocycler) which is more efficient

rapid- 100 billion copies of DNA can be made within hours

doesn't require living cells (such as bacterial cells)- quicker and less complicated techniques are needed compared to in vivo cloning

what are the disadvantages of in vitro cloning/ PCR

there are more errors in the DNA fragments produced compared to in vivo cloning

doesn’t synthesise products- unless DNA is incorporated into living cells

what are the advantages of in vivo cloning

there are less errors in the DNA fragments produced (with exceptions of mutations)

product synthesis- transformed bacteria can easily be cultured to produce the desired product e.g. insulin

what are the disadvantages of in vivo cloning

slower process (days/weeks to produce large quantities) than in vitro cloning which takes hours

uses living cells (bacterial cells) and vectors (plasmids)

what is gene therapy, and describe its use in medicine

gene therapy uses various methods to alter a persons genetic material

scientists are able to do this as they have a better understanding of the human genome and therefore, which genes may cause certain diseases

scientists can insert corrected copies of genes into patients to replace mutated genes to treat genetic diseases such as:

• cystic fibrosis

• severe combined immunodeficiency (SCID)

• cancer

how does a normal functioning CFTR channel differ in function to a mutant CFTR channel (cystic fibrosis)

normal functioning CFTR channel- moves chloride ions out of the cell

mutant CFTR channel- doesn’t move chloride ions out of the cell → water potential in the lumen remains low → water doesn't move out of the cell via osmosis → mucus remains viscous and sticky

what is cystic fibrosis caused by

a recessive mutated allele

the mutation is a deletion mutation that removes AAA (lysine)

what are the effects of cystic fibrosis

• thicker mucus in upper airways → increased risk of lung infection

• thicker mucus on alveolar surface → greater diffusion distance → reduced rate of gas exchange and O2 intake

• thick mucus can block pancreatic ducts → results in digestive enzymes not reaching the small intestine → poor digestion and absorption of nutrients

• thick mucus can block sperm ducts → infertility

what are two ways that gene therapy can be used to treat cystic fibrosis

gene replacement: mutated allele is replaced by a functional healthy gene (lysine won’t be removed → there will be functional CFTR channels

gene supplementation: one or more healthy copies are inserted. The genes added have dominant alleles so the are expressed over the recessive mutated alleles

what are the two types of gene therapy

somatic cell gene therapy

germ-line gene therapy

what is somatic cell gene therapy

somatic gene therapy targets the non-reproducing (somatic) cells (effects only affect the individual, not any offspring they may have)

effects are temporary as these cells are commonly replaced or may die → treatment must be repeated every few days

method:

a functional gene is inserted into the patients somatic cells using vectors such as viruses to deliver the gene into target cells (e.g. lungs for cystic fibrosis gene therapy)

the inserted gene will now produce the correct protein and the symptoms of the disease will be reduced or eliminated

give some examples of somatic cells

red blood cells

epithelial cells

lung cells (alveolar cells)

nerve cells

liver cells

muscle cells

what are some social and ethical issues with somatic gene therapy

lots of injections required (somatic cells die and are placed) → expensive treatment → not accessible to all people

people might become less accepting of disabilities as they become less common

doping in sports- genes in muscle cells are altered to increase EPO hormone production which promotes the formation of RBC’s

what is germ line gene therapy

it involves inserting functional genes into early embryos

the genetic change is permanent and heritable for both the individual and any offspring they may come to have themselves

it eliminates the faulty gene that may cause certain diseases

what are the ethical issues with germ line gene therapy and why is somatic cell gene therapy more ethical

can’t obtain consent from future individuals affected by the therapy (future individuals aren’t affected in somatic cell gene therapy as the changes aren’t heritable)

unknown long term effects can be passed onto descendants (changes aren’t heritable and passed on in somatic cell gene therapy

potential for misuse- such as designer babies (somatic cell gene therapy is generally limited to only the treatment of disease)

how can a virus vector be used to treat cystic fibrosis

uses an adenovirus (which is harmless) that is known to cause respiratory infections by injecting DNA into epithelial cells in the lungs

the adenovirus is modified so it can’t replicate

the adenovirus is cultured in epithelial cells alongside modified plasmids that contain the normal CFTR gene inserted

the CFTR gene in the recombinant plasmid DNA is taken up by some of the adenoviruses

the adenoviruses that have taken up the recombinant plasmid DNA are identified using markers and extracted from the epithelial cells

the modified virus is then introduced to the patients nostrils to hopefully produce the correct CFTR protein when they ‘infect’ the patient’s lung’s epithelial cells

this works because the virus will inject it’s own DNA containing the normal CFTR gene into the DNA of the epithelial cell, so the gene will be transcribed and translated to produce a normal functioning CFTR protein → thick and viscous mucus won’t build up as chloride ions are transported out of the cell normally

what is an issue with using viral vectors in somatic cell gene therapy

Viruses may mutate and cause infections

They might trigger an immune response so the virus is destroyed before being effective for treatment

Gene may be inserted into the genome that leads to a harmful genetic mutation- insertion mutation that leads to a different amino acid being encoded for

how can a plasmid vector be used to treat cystic fibrosis

uses in vivo cloning to produce transformed bacteria which contain recombinant plasmid DNA with the function gene

the plasmids are removed and wrapped in lipid molecules to produce a liposome

the liposome is administered to the patient’s nostrils using a nasal spray

the liposome moves into the patients epithelial cells in the lungs through the phospholipid membrane (because liposomes are lipid soluble), delivering the plasmid with the functional CFTR gene which will then be expressed → thick and viscous mucus won’t be produced as chloride ions are transported out of the cell normally

what is severe combined immune deficiency (SCID)

an inherited disorder

inability to produce mature T cells → can’t carry out the cellular response

inability to produce mature B cells → can’t produce antibodies involved in the humoral response

what are labelled DNA probes and DNA hybridisation used for

identifying and locating a particular length of DNA

to treat genetic diseases, the faulty gene that needs to be treated must be located

what are DNA probes

A DNA probe is a short length of single stranded DNA that has a known base sequence that is complimentary to the base sequence of a ‘known faulty/harmful allele’

the probe is attached to a radioactive or fluorescent label that indicates its position

how are DNA probes created

the DNA probe can be artificially synthesised using a gene machine (because we know the base sequence of the harmful allele)

how is single stranded DNA on the nylon membrane created, so that DNA probes can anneal

a cell sample is taken from a patient → can gather cheek cells using a swab inside of the patients mouth

The DNA is extracted from the cell sample and purified

the DNA sample is amplified using PCR

restriction endonucleases are used to shorten the DNA molecules (because the whole DNA molecules are too long to be analysed in one go)

the smaller DNA fragments are separated using gel electrophoresis

the negatively charged DNA fragments move through the pores in the gel towards the positively charged electrode

smaller DNA fragments are able to move at a faster rate through the pores, so they travel further

the DNA fragments separate according to size and charge producing bands in the gel

the bands of gel are transferred to a nylon membrane

the DNA fragments on the nylon membrane are single stranded- this is done by breaking the hydrogen bonds between complimentary base pairs

how are DNA probes and DNA hybridisation used together to indicate whether a specific harmful allele is present in a DNA sample

DNA probes are added to the nylon membrane

DNA probes have a specific base sequence that is complimentary to the harmful allele

the DNA on the nylon membrane is single stranded so the DNA probe can anneal to any complimentary strands

if the harmful allele is present in the DNA sample, then hydrogen bonds will form between the complimentary base pairs of the DNA strand and the DNA probe

before the DNA probes are identified the nylon membrane is washed to remove any excess DNA probes that haven’t formed hydrogen bonds (prevents false positive)

DNA probes with fluorescent label- UV light is used to detect their position

DNA probes with radioactive label- X-rays are used to detect their position

if the label shows up on the DNA fragments then the DNA contains the harmful allele

what is genetic screening used for

to see if you have alleles present in your genome for a particular genetic disorder

what is the process of genetic screening

Person’s DNA

a sample of DNA is taken from the person

the DNA sample is amplified using PCR

restriction endonucleases are used to shorten DNA molecule into smaller DNA fragments

DNA fragments are separated using gel electrophoresis → produces bands in the gel

the bands are transferred to a nylon membrane

the DNA fragments on the nylon membrane are single stranded- hydrogen bonds between complimentary bases are broken to form single strand from double strand

DNA probes

DNA probes are synthesised from a gene machine and have a complimentary base sequence to the allele causing the genetic disorder the person is being screened for

DNA probes are radioactively labelled

if the harmful allele is present in the single strand of DNA then the DNA probe will form hydrogen bonds with the complimentary bases and anneal to the strand

nylon membrane is washed to removed excess DNA probes that didn’t anneal

X ray films /autoradiography can then be used to detect radioactively labelled DNA probes that have attached to the strand of DNA → to tell whether the harmful allele is present in the persons DNA

what are the advantages of genetic screening

people can plan ahead about reproduction → might not want to pass on allele for disease to their children → can adopt instead

individuals can make lifestyle changes to reduce chances of developing the diseases (breast cancer)

these people can participate in research and clinical trials → help develop understanding and treatments of genetic diseases

what are some disadvantages of genetic screening

if the disease is incurable then it might cause the person anxiety and/or depression

the person might not actually develop the disease and would’ve still suffered from anxiety from thinking they might develop it

it could increase the price of someones life insurance- which they may not be able to afford

Why might allele's for diseases that develop later on in life be passed on in human populations

alleles are passed on through reproduction

symptoms might develop late in an individual → don’t know they have it before they reproduce and pass on the allele

describe the role of genetic counselling

used to help people understand and process their genetic screening results

they can also inform individuals of possible results before they have a screening

what may genetic counsellors discuss with a patient

the chances of the individual developing an inherited disease

the chance of an individual having a child that develops the genetic disease

the lifestyle changes that can be made to reduce the risk of developing the disease

possible treatments for the patients if they do develop the disease