DNA Technology

Making a protein using DNA technology involves a number of stages:

• Isolation of the DNA fragments that have the gene for the desired protein
• Insertion of the DNA fragments into a vector
• Transformation- tranfering the DNA into a suitable host cell
• Identification of thehost cells that have successfully taken up the gene by use of gene markers
• Growth/cloning of the population of host cells

Method 1: Reverse Transcriptase (Isolation)

• Many cells only contain two copies of each gene, making it difficult to obtain a DNA fragments containing the target gene, mRNA is therefore used instead as there are many more mRNA molecules complementary to the gene
• Retroviruses (e.g. HIV), unlike most organisms, are able to synthesise DNA from their RNA using an enzyme called reverse transcriptase, this enzyme is used to isolate the gene 
• 1. A cell that readily produces the required protein is selected (e.g. the beta-cells of the islets of Langerhans from the pancreas to produe insulin)
• 2. These cells have large quanties of the relevant mRNA, which is extracted
• 3. The reverse transcriptase is then used to make DNA from the RNA, this DNA is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA
• 4. To make theother strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene

Method 2: Polymerase Chain Reaction (PCR) (Growth)

Used to make millions of copies of a fragment of DNA

• 1. A reaction mixture is set up that contains the DNA sample, free nucleotides, DNA polymerase and primers- short pieces of DNA that are complementary to the bases at the start of the fragment you want
• 2. The DNA mixture is heated to 95 degrees C to break the hydrogen bonds between the two strands of DNA. The mixture is then cooled to 50-65 degrees C so that the primers can bind (anneal) to the strands
• 3. The reaction mixture is heated 72 degrees C, so DNA polymerase can work. The DNA polymerase lines up free DNA nucleotides alongside each template strand. Specific base pairing means new complementary strands are formed
• 4. Two new copies of the fragment of DNA are formed and one cycle of PCR is complete. The cycle starts again- the mixture is heated to 95 degrees C and this time all four strands (two original adn two new) are used as templates

Each PCR cycle doubles the amount of DNA e.g. 1st cycle- 2x2=4, 2nd cycle-4x2=8 ect.

Method 3: Restriction Endonuclease (part of Transformation)

• Some sections of DNA have palindromic sequences of nucleotides, these sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions)
• Restriction endonucleases are enzymes that recognices specific palindromic sequences (recognition sequences) and cut (digest) the DNA at these places
• Different restriction endonucleases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzyme's active site
• If the recognition sequences are present at either side of the DNA fragment you want, you can use restriction endonucleases to separate it from the rest of the DNA
• 1. the DNA sample is incubated with the specific restriction endonuclease, which cuts the DNA fragment via a hydrolysis reaction
• 2. Soemtimes the cut leaves two straight edges known a blunt ends, these are of no use, other times however the cut leaves sticky ends- small tails of unpaired bases at each end of the fragment
• 3. Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with the same complementary sequences

• Making lots of identical copies of a gene
• In Vitro cloning- where the gene copies are made outside of a living organism using PCR
• In Vivo cloning- where the gene copies are made within a living organism, as the organism grows and divides, it replicates its DNA, creating multiple copies of the gene
• Recombinant DNA- the DNA of two different organisms being combined
• GMO- a genetically modified organism

Part 1- Making recombinant DNA

The first step is to insert the DNA fragment into a Vector's plasmid, this is something that's used to transfer DNA into cell e.g. plasmids or bacteriophages (viruses that infect bacteria), in the case of a plasmid:

• The plasmid's DNA is isolated
• The plasmid's DNA is cut open in the middle of a gene that is resistant to anitobiotic A using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene, this means that the sticky ends of the plasmid DNA are complementary to the sticky ends of the DNA fragment conatining the gene
• The plasmid DNA and the DNA fragment are mixed together with DNA ligase, the enzyme joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA, the process is called ligation, the DNA fragment is now within the resistant gene of the plamid meaning it will no longer function
• The new combination of bases in the DNA is called recombinant DNA

*If the DNA fragment was made from mRNA or PCR, you'd need to incubate it with restriction enzymes to get the required sticky ends

Part 2- Transforming Cells

• The vector of the recombinant DNA is used to transfer the gene into cells- host cells
• Host cells that take up the vectors containing the gene of interest are said to be transformed
• If a plasmid vector is used, host cells have to be 'persuaded' to take in the plasmid vector and its DNA--> host bacterial cells are placed into ice-cold calcium chloride solution to make their cell walls more permeable, the plasmids are added and the mixture is heat-shocked (heated to around 42 degrees C for 1-2 minutes), which encourages the cells to take in the plasmids
• If a bateriophage vector is used, it will infect the host bacterium by injecting its DNA into it, the phage DNA (witht he targe gene in it) then integrates into the bacterial DNA

Part 3- Identifying Transformed Cells

Not all host cells will have taken up the vector and its DNA and so gene markers are used

• First it must be identified which bacterial cells have taken up the plasmid, the plasmids used contain two genes for antibiotic resistance, the first for anitbiotic A is non functioning due to the target gene being inserted in, the second gene for anitbiotic B is still functioning however adn so all the bacterial cells are grown on growth medium of anitbiotic B, those that survive contain the plasmid
• It must then be identified which host cells contain the plasmid with the new gene also, this uses the technique, replica plating, the cells that have survived are cultured on a agar plates, and a small sample of each colony is then placed in the exact same place as the original on a replica plate containing antibiotic A, the colonies that die must be the ones that have taken up the new gene as they are no longer resistant and the ones that survive contain only the plasmid
• Green flourescent protein (GFP)-transferred from a jellyfish with the target gene being placed within, the host cells that don't flouresce are the ones that have been transformed
• The enzyme lactase- the target gene is placed within the gene for the enzyme and those with the target gene will be unable to turn a particular substrate from colourless to blue

In Vivo cloning- advantages

• Can produce mRNA and protein as well as DNA due to it taking place in a living cell
• Can produce modified DNA, mRNA and protein- had modifications such as sugar and methyl groups added to them
• Large fragments of DNA can be cloned using in vivo cloning
• A realtively cheap method depending on how much DNA you want to produce
• No risk of contamination- the gene has been cut by the same restriction endonuclease which will match to the sticky ends of the opened up plasmid, contaminent DNA will therefore not be taken up by the plasmid
• Very accurate- many copies of a specific gene can be produced as opposed to a whole DNA sample

In Vivo cloning- disadvantages

• The DNA fragment has to be isolated from other cell components
• You may not want modified DNA
• Can be a slow process depending on how fast the bacteria grow
• Have to keep organisms alive

In vitro cloning (PCR)- advantages

• Can be used to produce DNA on a massive scale
• The DNA produced isn't modified
• Only replicates the DNA fragment of interest, isolation from a host DNA or cell component is not required
• A very rapid process
• Doesn't require living cells
• Automated

In vitro cloning (PCR)- disadvantages

• Can only replicate a small DNA fragment
• mRNA cannot be produced
• No way of expressing the gene to get the required protein e.g. insulin
• A risk of contamination- the sample must be very pure as unlike in vivo, any contaminent DNA will also be multiplied
• Expensive to produce a lot of DNA

• The manipulation of an organism's DNA
• Also known as recombinant DNA technology
• Transformed organisms- an organism that has had its DNA altered by genetic engineering
• Transformed plants- draught resistant, pest resistant, high crop yield
• Transformed animals- resistant to disease, produce lots of a desirable product e.g. milk

Substances produced by genetically engineered bacteria

• Antibiotics- produced naturally by bacteria which have been genetically engineered to produce greater quentities of these antibiotics at a faster rate
• Hormones- bacteria have been genetically engineered to produce human insulin, avoids killing animals and the issue of rejection from the individual's immune system
• Enzymes- many enzymes in the food industry are produced by genetically engineered bacteria e.g. amylase to break down starch during beer production

Agriculture

• Crops can be transformed so that they give higher yields or are more nutritious
• E.g. Golden rice- contains a gene from maize and a gene from a soil bacterium, which together enable the rice to produce beta-carotene which is used by our bodies to produce vitamin A. Golden rice is therefore produced in areas where there is a vitamin A deficiency e.g. Africa
• Crops can be transformed to be resistant to pests- fewer pesticides needed reducing costs and the amount of chemicals introduced into the environment
• Crops can be transformed to be drought resistant- survive in drought prone areas e.g. Africa

Industry

• Uses many enzymes as biological catalysts e.g. Chymosin is used in cheese making

Medicine

• Many drugs and vaccines are produced by transformed organisms using recombinant DNA technology, cheaper method making them more available to wider population

Agriculture

• A genetically identical monoculture would b vulnerable to disease
• Concerns over superweeds that would be resistant to herbicides as a result of transformed crops interbreeding with wild plants

Industry

• The issue of choice over whether you want to consume GM food or not
• Concerns over whether the prcoess used to purify proteins could lead to the introduction of toxins into the food industry

Medicine

• The process could be used unethically e.g. designer babies

Globally

• As this area advances the companies that own these methods will grow pushing smaller companies out of business

Cystic Fibrosis

• Caused by a mutant recessive allele in which 3 DNA bases (AAA) are missing- deletion
• ystic fibrosis trans-membrane-conductance regulator (CFTR) gene produces a non functioning protein as a result of the mutation
• CFTR is a chloride-ion channel protein that transports chloride ions out of the epithelial cells causing water to naturally follow by osmosis, in this way the epithelial membranes are kept moist
• In a patient with cystic fibrosis, the CFTR protein does not function properly and so the epithelial membranes remain dry and the mucus they produce remains viscous and sticky
• Symptoms include: high risk of infection due to mucus congestion, breathing difficulties due to a less efficient gaseous exchange, the formation of fibrous cysts due to the accumulation of thick mucus in the pancreatic ducts which prevents the pancreatic enzymes from reaching the duodenum and infertility due to the accumultion of thick mucus in the sperm ducts in males

• Gene replacement- the defective gene is replaced by a healthy gene
• Gene supplementation- one or more copies of the healthy gene are added alongside the defective gene, this is possible because the healthy gene is dominant over the recessive defective gene

Techniques

• Germ-line therapy- replacing or supplementing the defectiv gene in the ferilised egg, this ensures that all cells in the organism develop normally, as will all the cells of thei offspring. This is a permenant solution, affecting future generations, for this reason it is currently prohibited due to the long term genetic change
• Somatic-cell gene therapy- targets just the affected tissues, such as the lungs, a short term treatment due to the tissue treated constantly dying, the treatment must therefore be given on a frequent basis resulting in limited success

Delivering the Cloned CFTR Gene

The aim of somatic-cell gene therapy is to introduce cloned normal genes into the epithelial cells of the lungs, this can be carried out in two ways.

Using a harmless virus

• Adenoviruses cause cold and other respiratory diseases by injecting their DNA into the epithelial cells of the lung, they therefore make useful vectors for the transfer of the healthy CFTR gene
• 1. The adeno virsues are made harmless by interfering with the gene invloved with cell replication
• 2. The virus is grown in epithelial cells in the laboratory along with plasmids that have the CFTR gene inserted
• 3. The CFTR gene becomes incorporated into te DNA of the adenoviruses
• 4. The adenoviruses are isolated from the epithelial cells and purified
• 5. The adenoviruses are introduced into the nostrils of the patient
• 6. The adenoviruses inject their DNA, which includes the CFTR gene into the epithelial cells of the lungs

Wrapping the gene in lipid molecules

• Lipid molecules are used because can pass through the phospholibid of the cell surface membranes
• 1. CFTR genes are isolated from the healthy human tissue and inserted into the bacterial plasmid vectors
• 2. The plasmic vectors are reintroduced into their bacterial host cells and gene markers are used to detect which bacteria have successfully taken up the plasmids with the CFTR gene
• 3. These bacteria are cloned to produce multiple copies of the plasmids with the CFTR gene
• 4. The plasmids are extracted from the bacteria and wrapped in lipid molecules to form a liposome
• 5. The liposomes containing the CFTR gene are sprayed into the nostrils of the patient as an aerosol and are drawn down into the lungs during inhalation
• 6. The liposomes pass across the phospholipid portion of the cell-surface membrane of the epithlieal cells in the lungs

These forms of delivery ae not always effective because:

• Adenoviruses can cause infections
• Patients may develop immunity to adenoviruses
• The liposome aerosol may not be fine enough to pass though the tiny bronchioloes in the lungs
• Even where the CFTR gene is successfully delivered to the epithelial cells, very few are actually expressed due to the gene having to the enter the nucleus also

• Severe Combined Immunodeficiency
• Individuals do not show a cell mediated immune response nor are they able to produce antibodes
• The disorder arises when individuals inherit a defet in the gene that codes for the enzyme ADA, when functoning normally, this enzyme destroys toxins that would otherwise kill white blood cells
• Survival depends upon patients being raised in a strictly sterile environment of an isolation tent, or 'bubble', an giving them bone marrow transplants or injections of ADA

• The normal ADA gene is isolated from healthy human tissue using restriction endonucleases
• The ADA gene is inserted into a retrovirus
• The retroviruses are grown with host cells in the laboratory to increase their number and hence the number of copies of the ADA gene
• The retroviruses are mixed with the patient's T cells
• The retroviruses inject a copy of the normal ADA gene into the cells
• The T cells are reintroduced into the patient's blood to provide the genetic code needed to make ADA

The effectiveness of this treatment is limited due to T cells on living for 6-12 months. This means the treatment must constantly be repeated. More recent treatments have involved transforming the bone marrow stem cells which would give a constant supply of T cells.

Principles

• Not all of an individual's genetic material/genome codes for proteins, instead some of it consists of repetitive, non-coding base sequences
• The number of times these base sequences are repeated is different in from person to person resulting in the length of these sequences in nucleotides differing also
• The repeated sequences occur in lots of places in the genome, the number of times a sequence is repeated at different specific places (loci) in the genome can be compared between individuals- genetic fingerprinting

Step 1- PCR is used to make DNA fragments

• A sample of DNA is obtained e.g. saliva and PCR is used to make copies of the areas of DNA that contain the repeated sequences
• Primers are used that bind to either side of these repeats and so the whole repeat is amplified, different primers are used for each locus under investigation
• DNA fragments are produced where the length (nuclotides) corresponds to the number of repeats at a specific locus
• A flourescent tag is added (usually to the primers) so they can be viewed under UV

Step 2- Seperation of the DNA fragments by gel electrophoresis

• The DNA mixture is placed into a well in a slap of gel and covered in a buffer solution that conducts electricity
• An electrical current is passed though the gel
• The negative electrode is at the side with the well of DNA and the positive electrode is at the opposite end
• DNA is negatively charged, so it moves towards the positive electrode at the far end
• Shorter DNA fragments move faster and further through the gel, so the DNA fragments seperate according to length- a pattern of bands is produced

Step 3- Analysis of the genetic fingerprints

• The gel is placed under UV light where the DNA fragments can be seen as bands- genetic fingerprint
• A ladder may have been added on one as well- this is a mixture od DNA fragments of known length, allowing you to work out the lengths of the other bands in the gel
• Two genetic fingerprints can be compared to see if they match

• Determining genetic relationships- roughly half the non-coding base sequences are inherited from each parent, genetic fingerprinting can therefore be used in pternity tests as the more bands that match the more closely related you are. It can also be used on a more broader scale, for example looking at the relationship between to populations of organisms. It can also be used to trace only the male or female line of descent- for the female you look at DNA in the mitochondria and for the male you look at only the Y chromosome
• Determining genetic variability within a population- the greater the number of bands that don't match on a genetic fingerprint, the more genetically different the popualtion is
• Forensic science- used to compare the DNA taken from the crime scene and the DNA of suspects. If there is a match between the genetic fingerprints then the suspect can be linked to crime scene
• Medical diagnosis- can be used to diagnose genetic disorders an cancer, useful when the specific mutation isn't known because it identifies a broader, altered genetic pattern
• Animal and plant breeding- used to prevent interbreeding which can cause health problems, interbreeding decreases the gene pool leading to an increased risk of genetic disorders. It an also be used by animal breeders to prove pedigree of the animal

Locating Genes using DNA probes

• A DNA probe is a short single single strand of DNA that has specific bas sequence complementary to the target gene, his means that the probe will hybridise (bind) to the target gene if it is present in the sample
• The probe has a label attached so that it can be detected, the most common is radioactive label which can be detected by X-ray or a flourescent label which UV detects
• Step 1- a sample of DNA is digested into fragments using restriction enzymes and seperated using electrophoresis
• Step 2- the seperated DNA fragments are transferred to a nylon membrane and incubated with a flourescently labelled DNA probe, if the gene is present the probe with hybridise to it
• Step 3- the membraneis exposed to UV light and if the gene is present there will be a flourescent band

Gene sequencing techniques- restriction mapping

• Used to cut the gene into smaller sections so that it can be sequenced and then put back in order to that the entire sequence can be read in the right order
• Different ristriction endonuclease enzymes are used to cut labelled DNA into fragments
• The DNA fragments are seperated by electrophoresis
• The lengths of the fragments are used to determine the relative locations of the cut sites
• Once these are known, a restiction map of the original DNA can be made, this is a diagram of the piece of DNA showing the different cut sites, and so where the recognition sites of the restiction enzymes used to cut the DNA are found

Gene sequencing techniques- DNA base sequencing

Used to determine the order of bases in a section of DNA, so it can be used to sequence fragments of genes. It can be carried out by the chain termination method, which lets you sequence small fragments of DNA, up to 750 base pairs.

Step 1- a mixture of the following is added to four seperate tubes

• A single stranded DNA template- the DNA to be sequenced
• DNA polymerase- the enzyme that joins DNA nucleotides together
• DNA primer- short pieces of DNA
• Free nucleotides- A, T, C, G nucleotides
• A flourescently labelled modified nucleotide- once it is added to the DNA strand, no more bases are added after it (A*, T*, C*, G*)

Step 2- the tubes undergo PCR

• The strands are different lengths because each one terminates at a different point depending on where the modified nucleotide was added

Step 3- The DNA fragements are seperated using electrophoresis

• The fragments are visualised under UV light
• The complementary base sequence can be read from the gel
• The smallest nucleotide is at the bottom of the gel
• Each band after this represents one more base added
• By reading the gel upwards, the DNA sequence can be built up one base at a time

Modern DNA base sequencing

• This method can be done altogether in one tube in an automated DNA sequencer
• The tube contains all the modified nucleotide, each with a different coloured flourescent label
• A machine reads the sequence a produces and automated DNA sequence
• This is a much quicker and cheaper method, allowing whole genomes to be sequenced in a relatively small amount of time

If we know what the sequence of gene should be and what the mutated versions are, then we can use this information to screen people for a genetic disorder (screening- analysing a person's DNA to see if they have any mutations).

Sickle-cell anaemia

• A recessive genetic disorder caused by a mutation in the haemoglobin gene
• Causes and altered haemoglobin protein to be produced, which makes red blood cells sickle shaped, causing them to block capillaries and restrict blood flow
• People that are carriers are partially protected from malaria, this advantageous effect has caused an increase in the frequency of the sickle-cell allele in areas where malaria is common, therefore resulting in an increase in the likelihood of people in those area inheriting two copies of the allele and suffering from the disease

Screening for mutated genes

• DNA probes can be used to screen for clinically important genes
• To make a probe, the gene that you want to screen for is sequenced, PCR is then used to produce multiple copies of part of the gene- these are the probes
• Screening for a single gene- the probe can be labelled and used to look for a single gene in a sample of DNA (see 'locating genes using DNA probes')
• Screening for multiple genes- the probe can be used as part of a DNA mircoarray which can screen for lots of different genes at the same time. A DNA microarray is a glass slide with microscopic spots of different DNA probes attached to it in rows, a sample of human DNA with flourescent labels is washed over the array and any matching sequences will stick to the probes on the array. The array is then washed to removed any unattached DNA labels and the array is then visualised under UV light. Any spot that flouresces means that the person's DNA contains that specific gene

Developement of Scientific Techniques

• Techniques have evoloved to now be automated, more cost effective and efficient on a large scale
• For example screening for single gene at a time as evolved into being able to screen for multiple genes in any one time, this means medical diagnosis is getting faster and more precise

Uses of Screening

• Diagnosis and genetic counselling- genetic counselling is used to advise individuals who have a family history of a genetic disorder on screening and whether they want to be screened or not. If the result is positive then counselling is used to talk about prevention and treatment
• Deciding treatment options for cancer- Different mutations lead to different cancers, screening can therefore be used to identify specific mutation and then decide on the best course of treatment