GENE TECHNOLOGY

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Restriction enzymes

  • ezymes that cut DNA at specific sites
  • their proper name is RESTRICTION ENDONUCLEASES
  • some restriction enzymes cut straight across both chains forming blunt ends, most enzymes make a staggered cut forming sticky ends
  • they are called 'sticky' as they have short stretches of single stranded DNA
  • these sticky ends will 'stick' to other sticky ends by complementary base pairing (only sticky ends that are cut with the same restriction enzyme are complementary)
  • restriction enzymes have highly specific active sites and so will only cut DNA at specific base sequences 4-8 base pairs long (recognition sequences)
  • these are palindromic which means the sequence and its complement are the same but reversed (e.g. GAATTC has the complement CTTAAG)
  • the enzyme is produced natrually in bacteria as a defence to viruses (restrict viral growth)
  • they are enormously useful in genetic engineering for cutting DNA at precise places
  • short peices of DNA cut out by restriction enzymes are called restriction fragments
  • they are named after the bacteria they come from
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DNA ligase

  • an enzyme that repairs broken DNA by joining two nucleotides in a DNA strand
  • (a bit like DNA polymerase)
  • they're used in genetic engineering to do the reverse of restriction enzymes i.e. join together complementary restriction fragments
  • two restriction fragments can anneal if they have complementary sticky ends, but only by weak hydrogen bonds
  • the backbone is incomplete
  • it is DNA ligase that completes this backbone
  • restriction enzymes and DNA ligase can be used together can be used to join lengths of DNA from different sources
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Reverse transcriptase

  • this enzyme does the reverse of transcription: synthesises DNA from RNA templates
  • it is produced natrually in a group of viruses called retroviruses (it helps them invade cells)
  • used to make 'artifical genes' called complementary DNA (cDNA) from mRNA
  • mRNA (without introns) is extracted and mixed with reverse transcriptase (RT) and DNA nucleotides
  • a new strand of DNA is synthesised complementary to the mRNA, forming a double stranded DNA/RNA molecule
  • the strands are then seperated and RT synthesises a second strand of DNA complementary to the first
  • the result is a normal double helix strand of DNA called cDNA  (cDNA is shorter as it has no introns)

uses of RT in gene technology:

  • make genes without introns that can be expressed in bacteria
  • make a stable copy of a gene (RNA is more readily broken down than DNA)
  • makes genes easier to find, there are 1000's of genes in a human genome, but a given cell only expresses a few genes, so only makes a few kinds of mRNA (which can be used to make cDNA)
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Electrophoresis

  • a form of cromatography used to seperate DNA fragments on the basis of their length (typically restriction fragments)
  • DNA samples are placed in wells at one end (-) of a thin slab of gel and covered in a buffer
  • an electrical current is passed through the gel
  • each DNA nucleotide contains a negatively charged phosphate group, so the DNA is attracted to the positive electrode
  • the gel adds restriction so the longer lengths of DNA are more restricted than the shorter lengths
  • thus, the shorter lengths move further towards the positive electrode

The DNA cannot be seen on the gel so they must be visualised, there are 2 common methods:

  • 1) stain the DNA witha flourescent marker which emits light when illuminated with UV light
  • 2) label the DNA samples with a radioactive isotope, which allows them to be visualised via autoradiography (DNA shows up as dark bands)
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DNA sequencing

  • this means reading the base sequence of a length of DNA using the sanger method

Method:

1) label 4 test tubes A, T, C and G and add:

  • -a sample of DNA to be sequenced
  • -a radioactive primer (so the DNA can be visualised later on in the gel)
  • -4 DNA nucleotides (A, T, C and G)
  • -DNA polymerase (synthesise many copies of DNA)
  • -modified/terminator nucleotide (stops further synthesis of DNA, add A nucleotide to tube A etc.)

2) let DNA polymerase synthesise many copies of the DNA samples, at random a modified nucleotide will be added and stop further synthesis (all fragments in tube A will be stoped at A etc.), a range of DNA molecules will be made ranging from full length to very short

3) run the contents of the 4 tubes side by side on an electrophoresis gel, visualise the DNA using autoradiography, since the fragments are now sorted by length the sequence can be read of the gel, starting with the smallest fragment (read from the bottom upwards)

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Automation of DNA sequencing

  • most DNA sequencing now carried out by machines
  • flourescently labelled dyes used by the machine rather than radioactive markers
  • due to each modified nucleotide having one colour assiociated with it the whole process can be done in one test tube
  • PCR cycles are used to speed up the process
  • the results from electrophoresis are scanned by lasers and interpreted by computer software
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Restriction mapping

  • a restriction map is simply a diagram of a piece of DNA marked with the locations of sites where it is cut by restriction enzymes
  • EXAMPLE:
  • a piece of DNA is cut with two different restriction enzymes, both on their own and as a pair, this gives three different mixtures of restriction fragments which are run on electrophoresis gel
  • the first lane on the gel contains a 'DNA ladder' - a mixture of DNA fragments of known sizes
  • comparison to the ladder allows us to meause the length (in kb) of each fragment
  • firstly add the fragments lengths up e.g. E2 produced 7 and 10 and E1 produced 8, 6 and 3 so the overal length of the DNA is 17
  • next work out the number of recognition sites e.g. E1 produced 3 fragments so must have 3 recognition sites, E2 produced 2 fragments so must have 1 recognition site
  • by the process of logic you should be able to map out where the enzymes cut on the piece of DNA

The map is useful because:

  • it can be used to choose restriction enzymes to generate known sized fragments
  • it can also be used to aid DNA sequencing as shorter fragments are easier to sequence
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Polymerase chain reaction

  • used to copy DNA samples in a test tube (in vitro)
  • 1) sample of DNA to be copied, all 4 nucleotides and the enzyme DNA polymerase are added to a test tube
  • 2) the sample is heated to 95 degrees( to break the hydrogen bonds and seperate the two strands)
  • 3) the sample is cooled to 40 degrees (so hydrogen bonds can form) and primers are added-primers are short lengths of DNA that anneal to complementary sequences on the two strands forming short sequences of double stranded DNA, this is done for 2 reasons:
  • the enzyme DNA polymerase requires some double stranded DNA to get started
  • only DNA between the primers is replicated - choosing appropriate primers ensures only a specific target sequence is copied
  • 4) DNA polymerase can now build new strands on the old strands to make double stranded DNA (this is done through complementary base pairing) - the enzyme used in the PCR is derived from bacteria from hot springs and has an optimum tempreture of 72 degrees (this allows replication to occur quickly)
  • 5) each original DNA molecule is replicated to form 2 molecles - the cylce is repeated from step 2 and each time the number of molecules of DNA doubles (hence why it is called a chain reaction) - typically the cycle is repeated 20-30 times which produces over a million molecules
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DNA probes

  • a DNA probe is simply a short length of single stranded DNA with a label attatched
  • there are two common types of label:
  • 1) radioactively labelled - visualised using autoradiography
  • 2) fluorescently labelled - visualised using UV light
  • probes can be made to flouresce with different colours
  • a probe is added to a single strard of DNA (e.g. a DNA fragment)
  • they are usually made to anneal with a specific sequence of bases - in order to do this we need to first know the DNA sequence
  • complementary probes and sections of DNA condence to form a hybrid peice of DNA (DNA from two different locations) via a process called hybridisation
  • the site at which the probe binds is identified by its label mentioned above
  • USES:
  • identify restriction fragments containing a paticular gene out of thousands of restriction fragments from a whole genome
  • identify genes in one species that are similar to those in another
  • to identify short sequences of DNA used in DNA fingerprinting
  • to screen for genetic diseases e.g. huntingtons and cystic fibrosis
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Southern blotting

  • an electrophoresis gel cannot be stored or manipulated as it is too fragile and the seperated DNA fragments continue to diffuse through it (blurring the bands)
  • southern blotting is used to overcome this, the process is as follows:

1) a thin nylon membrane is laid over the gel

2) the membrane is covered with several sheets of absorbent paper, which draws up the liquid containing the DNA by capillary action

3) this transfers the DNA fragments to the nylon membrane in precisely the same relative positions they occupied on the gel

4) DNA fragments are then fixed to the nylon membrane by UV light

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Genetic fingerprinting

  • used to distinguish between different peoples DNA samples
  • based on the fact that the genome of an organism is made of many repetetive non-coding bases of DNA (around 95% does not code for a characteristic)
  • these non coding bases are called introns and they contain repetitive sequences called core sequences
  • these sequences are of different numbers and length in different individuals
  • more closely related two individuals are the more similar their core sequences will be
  • process has 5 main stages: extraction, digestion, seperation, hybridisation, development (Elephants Dont Share Hot Dogs)
  • 1) EXTRACTION - the tiniest sample of tissue is enough for genetic fingerprinting, seperate the DNA from the rest of the cell, increase quantity using the PCR
  • 2) DIGESTION - DNA is cut into fragments by restriction endonucleases, they are chosen for their ability to cut just outside, but not within, the core sequences
  • 3) SEPERATION - fragments are seperated according to size by gel electrophoresis, the gel is then immersed in alkali to seperate the double strands, single strands are transferred to a nylon memrane via southen blotting

turn over for genetic fingerprinting cont.

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Genetic fingerprinting cont.

  • 4) HYBRIDISATION - radioactive (or flourescent) DNA probes are used to bind with core sequences, probes have base sequences that a complementary to the core sequence, they condence under specific conditions e.g. temp and ph, this process is carried out with different probes for different core sequences
  • 5) DEVELOPMENT - xray film placed on the nylon membrane and the film is exposed to the radiation from the radioactive probes (if flourescent probes used the positions are located visually under UV light), the pattern of bands formed is unique to each individual apart from identical twins
  • INTERPRETING THE RESULTS:
  • DNA fingerprints from two samples e.g. blood at a crime scene and blood from a suspect are visually checked
  • if it appears to be a match the patterns of bars from each sample are passed though an automated scanning machine
  • this machine calculates the length of DNA fragments by measuring the distances travelled in electrophoresis by known lengths of DNA
  • finally, the odds of someone else having an identical fingerprint are calculated
  • the closer the match between the two pattern the greater the probability they are from the same person
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Uses of genetic fingerprinting

  • in forensic sciences, to match DNA samples collected from a crime (blood, hair etc.) with that of suspects
  • determine family relationships, usually between a child and a suspected father (paternity test) - since children inherit 50% of their DNA from their father, half their DNA fingerprint should match his
  • to prevent undesirable interbreeding in farms and zoos
  • identify plants and animals that carry desirable or undesirable alleles
  • determine relationships between ancient humans (DNA extracted from archaeological remains) and modern humans
  • establish evoloutionary relationships between different species
  • measure genetic diversity within a population
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Vectors

  • a vector is a length of DNA that carries the gene we want into the host cell
  • its needed because a length of DNA containing a gene wont do anything inside a host cell
  • since its not part of the cells normal genome it wont be replicated when the cell divides, it wont be expresssed and would be broken down quickly
  • a vector gets around these problems by having the following properties:
  • 1) its big enough to hold the gene we want, but not too big
  • 2) its closed loop so is less likely to be broken down
  • 3) it contains control sequences e.g replication origin so that the gene will be replicated, expressed or incorporated into the cells normal genome
  • 4) contains marker genes so that cells containing the vector can be identified
  • PLASMIDS are the most common type of vertor
  • they are found in bacterial cells
  • plasmids are copied seperately from the main bacterial DNA during replication so the plasmid genes are passed on to all daughter cells
  • foreign genes are easily incorporated into plasmids using restriction enzymes and DNA ligase
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Inserting a gene into a plasmid

  • 1) restriction enzyme used to cut the gene from the donor DNA with sticky ends
  • 2) the SAME restriction enzyme cuts the plasmid in the middle of one of the marker genes
  • 3) the gene and plasmid are mixed in a test tube and they anneal because they where cut with the same restriction endonuclease and so have the same sticky ends
  • 4) the fragments are joined covalently with DNA ligase to form a hybrid vector (hybrid of bacterial ad foreign DNA)
  • 5) several products are also formed: some plasmids re-anneal with themselves and some DNA fragments form chains or circles
  • 6) these products are hard to seperate but this doesnt matter as the marker genes can be used to later locate the correct hybrid vectors
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Transformation

  • means inserting DNA (in a plasmid) into a living cell (host cell) - the host cell is therefore genetically motified or transformed
  • a transformed cell can replicate and express the genes in the new DNA
  • however, DNA is a large molecule and doesnt readily cross the cell membrane, so the membrane must be made permeable in some way
  • the way this is done depends on the type of host cell
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Marker genes (cells who do and dont have the plasm

  • marker genes are used to find which cells have taken up the plasmid with the gene (hybrid vector)
  • following transformation there are 4 possible outcomes
  • 1) no DNA taken up
  • 2) foreign DNA taken up
  • 3) original plasmid taken up
  • 4) hybrid plasmid taken up
  • vectors contain two different marker genes:
  • the first marker gene distinguishes between those cells who have taken up a plasmid and those who havent
  • the plasmid contains a gene for resistance to an antibiotic e.g. ampicillin
  • bacterial cells that take up the plasmid therefore make this gene product and are resistant
  • the cells are grown on a medium containing ampicillin, which kills all the untrasformed cells
  • only those who are transformed survive - these are transferred to another plate
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Marker genes (plasmids with gene and without)

  • second marker gene distinguishes between cells that have taken up the hybrid plasmid and those who have taken up the original plasmid
  • the trick is, the foreign DNA was interted INSIDE the marker gene, so the cells with the hybrid plasmid CANNOT make their gene product
  • different genes are used for this second marker:

1) gene for resistance to another antibiotic e.g. tetracycline - cells with hybrid are not resistant, since this means killing the cells we want the test is done on a replica plate, colonies of each cell grow and a small proportion of these colonies is transferred to the replica plate, colonies that grow on the first plate (ampicillin) and not the second plate (tetracycline) are the ones with the hybrid vector and hence the ones we want

2) gene for an enzyme e.g. lactase - this enzyme turns white substrate in the agar plate blue, so colonies with the original plasmid turn blue and those with the hybrid plasmid remain white and are easily identified

3) gene for a green flourescent protein (GFP) - colonies with the original plasmid flouresce green, those with the hybrid vector do not flouresce and so are easily identified

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Gene cloning (in vitro and in vivo)

  • gene cloning is simply making multiple copies of a piece of DNA
  • necessary step in just about any aspect of gene technology e.g. engineering, sequencing, fingerprinting
  • 2 different types:
  • in vitro ( in glass) - uses PCR in a test tube (newer technique)
  • simple, automated technique, complete in few hours
  • clones DNA molecules up to 1kbp long
  • very sensitive, can clone a single molecule
  • can use DNA from different kinds of source e.g. crime scenes
  • high error rate, no error correction
  • DNA is made in the test tube so cannot be expressed directly
  • in vivo (in life) - uses living cells such as bacteria (older technique)
  • complex, multistage process, several days to complete
  • clones DNA up to 2Mbp long
  • low error rate due to error correction mechanisms
  • large amounts of original DNA needed
  • DNA made in cells, so can be expressed easily
  • need intact, pure DNA
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Use of recominant DNA technology

  • producing a genetically modified organism (GMO) with a new genotype
  • we'll consider the application in 3 groups:
  • 1) Gene products - using GMO's to produce chemicals for medical or industrial applications
  • 2) New phenotypes - gene technology to alter the characteristics of an organism (usally farm animals or crops)
  • 3) Gene therapy - using gene technology on humans to treat disease

gene products examples:

  • hormones such as insulin, HGH, BST
  • enzymes such as AAT and DNAase both used to treat cystic fibrosis, rennin and cellulase
  • antibiotics (improved quality, treat disease faster)

new phenotypes examples:

  • PLANTS - long life tomatoes, insect/herbicide/pest resistant crops, nitrogen fixing crops
  • ANIMALS - cattle resitant to certain diseases, tick-resistant sheep, fast growing fish
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Evaluating gene technology

Think MORALS, ETHICS, SOCIAL ISSUES

  • medicines and drugs can be produced safely in large quantities from microbes rather than slaughtered animals
  • agricultural productivity improved while using less pesticides and fertilisers, so helping the enviroment
  • GM crops improve the nutrition and health of millions of people
  • we can make plants more tolerent of enviromental extremes
  • help prevent certain diseases e.g. rice can be given the gene for vitamin A (vitamin A deficiency causes blindless in thousands of people (ethically wrong to prevent this?)
  • can cure certain genetic disorders e.g. cystic fibrosis
  • plants can be modified to produce antigens that can be inserted into humans to induce natrual antibody production
  • microorganisms can be used to control pollution e.g. clean up oil slicks
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evaluating gene technology cont.

  • risk to modified organism - e.g. cancer and metabolic diseases
  • transfer to other organisms - e.g. the gene for herbicide recistance being transferred from a crop to a weed
  • risk to the ecosystem - could affect its delicate balance and food webs
  • risk to biodiversity - continue to reduce genetic diversity thats already occured as a result of selective breeding
  • risk to human societies - unexpected and complicated social and economic consequences e.g. bananas could be grown in temperate countries which would have disastrous effects on economies such as the caribbean that rely on money from these exports
  • risk to local farmers - ownership of gene technology remains with big corporations, benefits are not available to farmers in third world countries who need it the most
  • is it immoral to tamper with genes at all?
  • we can not be sure of the long term effects on things such as evoloution
  • what are the consequences of the ability to manipulate genes getting into the wrong hands
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Gene therapy - methods

  • the correct gene needs to be introduced into human cells where it can be expressed
  • some of the most common methods are:
  • 1) LIPOSOMES - the correct gene is incased in a lipid vesicle called a liposome, the liposome membrane fuses with the cell mebrane, delivering the correct gene to the cell
  • 2) VIRUSES - normal viral infection depends on the virus delivering its own DNA into the host cell where it can be expressed, so genetically modified viruses can be used to deliver human genes by the same method, the virus must first be genetically engineered to make it 'safe' so it can't reproduce or make toxins
  • 3) STEM CELLS - in some cases stem cells can be removed from the patient e.g. from bone marrow, it can be genetically modified in vitro with the correct gene then injected back into the patient, this is safer as it prevents immune rejection

NOTE: gene therapy doesnt alter or replace the existing mutated gene, but in addition the new gene will make a working protein and allow the modified cells to function properly

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Gene therapy - examples

CYSTIC FIBROSIS

  • the most common disease in the UK (affecting 1 in 2500 people)
  • caused by a mutation in the gene for the protein CFTR, which is a chloride ion channel
  • this results in CL- being retained in epithelial cells, lowering their water potential, meaning water does not move out by osmosis and mucus surrounding the cells is viscous and sticky
  • this results in a number of problems including breathlessness, lung infections etc.
  • CF is always fatal but if around 10% of the epithelial cells could be modified then it would allow enough chloride ion transport to relieve symptoms
  • in clinical trials lisosomes and viruses are delivered using an aerosol inhaler - no therapy has been shown to be successful

SCID (SEVERE COMBINED IMMUNODEFICIENCY DISEASE)

  • very rare genetic disease that affects the immune system
  • mutation of the gene for the enzyme ADA, without this enzyme wbc cannot be made, so sufferers have almost no effective immune system so have to spend their lives in sterile isolation - recent gene therapy has proved effective though
  • bone marrow cells are tranfected with the ADA gene in vitro then injected into the patient
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Gene therapy - problems

  • most gene therapy attempted so far has a short lived effect - problems with integrating the new gene in the host cell and replicating new DNA with the gene in it once in the host cell mean patients have to repeat the therapy at intervals
  • there is a chance that the new gene may be recognised as non self - this would lead to it being destroyed in a primary respose, constant repeated treatment stimulates a greater secondary response which could be harmful
  • theres a chance the new gene could be incorporated into the host in the middle of another gene e.g. a tumour supressor gene - therefore gene therapy could induce cancer, this occured in a clinical trial of SCID where 3 in 20 patients developed leukaemia
  • viruses are the most successful vectors in gene therapy but they present a variety of potential problems e.g. toxicity, immune and immflammatory responses - in 1999 a gene therapy patient died of a massive immune response after a virus was used
  • common genetic disorders e.g. heart disease, high BP, alzheimers disease, arthiritis and diabetes are all caused by the combined effects of many genes (multigene disorders) and are impossible to treat with gene therapy
  • the genes are not always expressed
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Gene therapy - types

  • important to appreciate the difference between somatic gene therapy and germ-line gene therapy

SOMATIC GENE THERAPY

  • genetically altering specific body cells e.g. epithelial cells on the trachea, bone marrow cells, pancreas cells in order to treat a disease
  • it will potentially treat the disease in the patient but any genetic changes will NOT be passed on to the offspring

GERM-LINE GENE THERAPY

  • genetically altering those cells (sperm, ova, zygotes, early embryo cells) that will pass their genes down the 'germ-line' to future generations
  • this would affect every cell in the resulting humans and in all his/her decendents
  • germ-line therapy would be very effective but is also potentially dangerous (since long term effects are unknown), unethical (easily leads to eugenics), immoral (since is could involve altering or destroying human embryo's)
  • it is currently illegal in UK and other countries (apart from in animals)
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Genetic screening

  • involves testing for the presence of clinically important alleles - this can be used to select appriopriate treatment at early stages or, if uncurable, help in family planning and care
  • procedure:
  • 1) the order of nucleotides on a mutated gene is determined by DNA sequencing, genetic libraries now store genetic sequences of many genes reponsible for genetic diseases
  • 2) fragment of DNA made that is complementary to the mutated portion of the gene
  • 3) DNA probe is formed and labelled (flourescently or radioactively)
  • 4) PCR techniques produce multiple copies of the DNA probe
  • 5) probe is added to a single strand of DNA from the person being screened
  • 6) if the person has the mutated gene it will have bases complementary to the probe, the probe will anneal to the single strand
  • 7) the DNA fragments will now be labelled with the probe and so can be distinguished from the other fragments by the use of autoradiograpghy and an xray film or a UV light
  • 8) if complementary fragments are present then they will either be taken up by the xray film or flouresce
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When genetic screening is done

  • ADULT SCREENING
  • tests adults for alleles of late-onset diseases such as huntingtons or breast cancer before any symptoms appear
  • also used to test for carriers of a genetic disease e.g. cystic fibrosis
  • NEWBORN SCREENING
  • immediately after birth a blood sample is tested
  • DNA is extracted from the baby's cells and tested for alleles for conditions that benefit from being treated in early life
  • PARENTAL SCREENING
  • carried out on fetal cells before birth, offerered when there is a high risk the fetus may have a serious genetic disability
  • DNA from these cells are tested for alleles for diseases such as tay sachs and sickle cell anemia
  • this gives parents the option to terminate the pregnancy or prepare for care of a disabled child
  • PRE-IMPLANTATION SCREENING
  • performed on human embryos created by IVF, 1 or 2 cells are carfully removed from the 8-cell embryo and the DNA is tested - the procedure doesnt harm the embryo
  • embryo's without genetic disorders are used for implantation
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Genetic councelling

  • results of genetic screening can be complex and distressing and so are often accompanied by genetic counselling
  • a genetic counsellor can explain the meaning of a test result, discuss the implications for the patient and their families and advise on the next course of action
  • SICKLE-CELL ANEMIA - caused by a recessive mutation on the Hb gene, a woman may be a carrier (it will not affect her) but theres a 25% chance her child could suffer if her partner is a carrier too, a genetic counsellor can discuss the option of pre-implantation/pre-natal screening
  • HUNTINGTON'S - caused by a dominant allele so a positive result means they will develop huntingtons, causes muscle spasms, death and occurs in middle age (no cure), genetic councelling is very important in this case, 95% of individuals at risk choose not to get tested
  • CANCER - gene probes have been made for hundreds of cancer-related alleles, screening and councelling can help choose the best treatment for the patient early on

Problems with genetic screening -

  • mistakes in the procedure and interpretation lead to false positives and false negatives
  • since genetic tests must remain confidential they can lead to genetic discrimination when applying for things like insurance or a job
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