Genomes and Gene Therapy

Genomes and Gene Therapy

1. A/G/H) Sequencing a Genome

   • 1. PCR
     • In PCR machine = DNA strand, Nucleotides, DNA Poly in buffer solution 1. DNA denatured & strands seperate (95) 2. Annealing - add Primer (starting point of DNA snythesis, complementary to beginning of DNA) is added  (65) 3. DNA Polymerase adds free nucleotides to exposed base pairs starting at primer (72 evolved in hot climate) 4. Heat again = 4 from 2 and repeat   
     • For the above process (Sanger) to work, the target DNA needs to be mass produced: in order to generate enough DNA fragments by Sanger sequencing to be able to decipher a genetic code, millions of copies of the original DNA sequence must be produced. This can be done by a process called polymerase chain reaction (PCR) 
     • Whole DNA
     • Artifical DNA replication
     • 95, 65, 72
   • 2. Fragmentation OR restriction enzymes
     • Fragmentation High Pressure Small Cappilary Tube  Breaks into fragments
     • Restricition Enzymes - Recognise specific Paliodromic  sequneces (read the same in oppostie directions)  - Different RE for different Sequence - Active Site specfic  - Incubate with DNA &  cuts via hydrolyses reaction - cut leave sticky ends, (single exposed bases at end), complementary to other DNA cut by same RE  - blunt ends & sticky ends added 
   • 3. Multiple labeled copies of genome (Pretty much PCR again but called Sanger Sequencing)
     • 1. Primer Added 2. DNA Polymerase added 3. Normal Nucleotides added & Labeled nucleotides added  - Labeled using flurecent die (1 die for each base) & double deoxidised (lack hydroxyl group) so stop DNA poly adding nucletides once has joined to DNA strand 4. Multiples copies made of particular sections of DNA, not full sections as stop at labeled nucleotide = Many different chains all with different lengths ending in with Labeled nucleotide 5. repeated many times useing same template strand of DNA   
     • To decode a sequence of DNA, the template DNA is copied repeatedly, with the copies terminating at different points. The copies can then be arranged into order of size to determine the sequence. 
     • Primer
       • starting point of DNA snythesis, comp to beginning of DNA
     • Labeled Nucleotides
       • Double Deoxides = STOP DNA polymerase  Fluourese 
     • DNA Polymerase
     • DNA Fagments
   • 4. Electropherises
     • Just electropherises not agrose gel through small capillary tube time taken to reach certain point
     • Exam Q
       • explain why the position of allele 2 on the electrophoresis gel indicates that it contains a deletion in comparison with allele [3] (ii) deletion removes base pairs; shorter/lighter, pieces of DNA move further in electrophoresis; towards anode; so allele 2, shorter/lighter, than allele 1;    
   • Gene Sequencing
     • tagging an entire length of dna, a gene using a dna probe
     • DNA Probes
       • DNA probes added to locate DNA (Probe labeled with Flurocence/radioactive)
     • Agrose Electropherisis
       • Used for genetic finger printing and identifying a gene 1. tank set up containg Agrose Gel submerged in buffer solution 2. Dna fragments added 3. Voltage applied across gel using 2 anodes 4. DNA nucletodies have phosphte so attracted to Postive anode 5. Small (measured in bases)move faster, Big move slower 6. Labeled nucletide recorded when pass a particular point/ time take to get to particlar point/distance moved in specific time 6. Turn off voltage and different sized fragments in different areas 7. make visible when moved onto absorbent paper & heated = DNA strands sperate 8. DNA probes added to locate DNA (Probe labeled with Flurocence/radioactive) 9. Shine UV light/on X ray film = see DNA 

2. B) Genome Sequencing = Genome wide Comparisons

   • 1. Can be compared between organisms of DIFFERENT species
     • Medical Research
       • -Carry out medical research - human genes cause disease found in other animals that can then be used to test medicine for disease on 
     • Development
       • - Help understand development and how its controlled e.g. Gene seq revealed homeobox genes in Animal, Plants & Fungi
     • Evol Relationships
       • -All shared Common Ancestor  Closely related species evolved away from each other  DNA tell us how closely related 2 species are (humans & Chimps)
   • 2. & SAME Species
     • Human Migration
       • During evolution humans have moved to different parts of the world we can use gene sequencing to see how this effects their genome  = build up a picture of early human migration
     • Study Genetics of Disease
       • Mutations can lead to disease - by comparing suffers we can see which mutation carries highest severity & between sufferers and non sufferers = what and where mutation is 
     • Medication
       • Comparing genomes on medication could show us which genome is responding best to particular medication could be possible to sequence genome to specific medication (or vice versa = more effective 

3. C) Define Recombinant DNA

   • DNA altered by Genetic engineering  Contains Lengths from two different species  "DNA formed by joining together two different sources"
   • Exam Q: "Outline formation of Recombinant DNA"
     • 3Marker Perfect Answer: DNA altered by genetic engineering and contains two lengths of DNA from different species  Cut using restriction enzymes  = Complementary sticky ends  DNA Ligase seals nicks in sugar phosphate backbone 

4. D/E) Explain Genetic Engineering

   • Extraction of gene from one organism
     • 1. Identify
     • 2. Cut OR made
       • -using Restricition enzymes (RE) (made by bacteria) or DNA made by reverse transcriptase of MRNA  - RE specfic paliodromic DNA so need to have particular base seq  -cut two strand at diferent points = sticky ends 
       • If gene very small but have high proportion if mRNA can be made (insulin) 1. mRNA extracted  2. Incubated with reverser transcriptase (retro virus = makes DNA from RNa) 3.Comp single stranded DNA made from RNA  4. Sticky ends added by placing single standed ends of particular base on gene and comp bases added to vector transfering gene into organism 
     • 3. Copied
       • using prc (explained in A)
   • Def: Use gene technology to change genetic materal of an organism..
     • can invove taking genes from one organism and placing in other so gene is expressed. Organism altered by process = transformed organism  Org enginered to express gene from different species = transgenic organism
     • Transgenic Org
       • Org with expressing dna from DIFFERENT species 
     • Transformed Org
       • Orgs altered by genetic engineering

5. F) Dna Frag seperated using Electropherises

   • Explained in A  2 different types cappilary tube = time taken = for genome gene sequencing = agrose gel = distance traveled

6. G/H) Probes & PCR

   • Explained in D/E & A  Probes used for sequencing a gene

7. I) Gene in to Vector (plasmid)

   • Vector
     • Plasmid is commonly used vector Made naturally by bacteria  small circular DNA often contain antibitotic resistanse gene making it easy to identify which gene has taken up plasmid are exchanged between bacteria 
   • Inserting
     • 1. bacteria treated with enzymes/ 2. Centrifuged to sperate large chromosomal dna and plasmids 4. Plasmids selected and cut using same RE used to cut gene = comp sticky ends. Plasmids and Gene mixed together & some will pair up.  6.Ligase is used to seal nicks in sugar phosphate backbone = ligation 7. Some plasmids now contain specific gene = recombinant plasmid/dna 
   • Exam Q
     • (3)state 3 ways in which the structure of plasmid differs from structure of protein  nucleotides / sugar + phosphate + base ; amino acids ; 4 different subunits ; 20 different subunits ; phosphodiester bonds ; A phosphoester peptide bonds / polypeptide ; contains P ; contains S / disulphide bonds ; double-stranded / double helix ; may have 4º structure ; circular ; ref to, 2º / 3º, structure / AW ; AVP ; e.g. role of H bonds 3 max   

8. J) Other Vectors

   • used to transfer the gene into bacterial cells 
   • Viruses
     • They can divide in cell and Insert dna into cell often called DNA probes added to locate DNA (Probe labeled with Flurocence/radioactive) they work by injecting their DNA into cell
   • Liposomes
     • are lipid soluble so go straight through the plasma membrance  they are tiny balls of lipid containg dna 

9. K) Bac take up Plasmid

   • 1. Mixing
     • 1. Bacteria + Calcium Chloride = Bacteria cells walls permable to plasmids  2. Bacteria + Calcium + Plasmid (Heat shocked to 42 for 2 mins = encourage uptake of plasmid)  and take up plasmids BUT not sure which plasmids are recombinant/not
   • 2. Who has taken up who?
     • To identify which bacteria have taken up the recombinant plasmids we use their antibiotic resistance to identify them Plasmids can contain one gene that makes the resistant to tetracycline and one gene that makes the resistant to ampicillian & if speciic RE used to cut the plasmid cuts through the tetracycline resistance gene = not work. SO plasmids that are Recom are resistance to ampacillian but not tetracycline 
     • 1. bacteria grown or agar jelly that cointains ampicillin
       • Any that survive must have taken up A plasmid  so this eliminates bacteria that hasn't take up a plasmid.
     • 2. Sample from each colony Moved to agar with Tetracycline
       • moving to lots of different agars = replica plating 1.Moved using a sterile cloth that touches the master plate and then the tetracycline plate  2. Then icubated 3. Those that grow contain plasmids that have not go recombinant gene 
     • 3. Go back to master plate and identify colonies not on 2 plate
       • These are the colonise that are not resistant to tetracyline but have taken up a plasmid so must be the Recom plasmid 
   • 3. Recomb Bac now grown
     • On large scale in fermenter where they reproduce 
   • Exam Q
     • Suggest two ways in which the bacteria which take up the modified plasmids can be idetified Identifying bacteria using antibiotic reistants or adding a fluorescensent tag that shines under UV light

10. L) Describe advantages of Microorg taking up Plasmids

    • Can take up from enviro
      • This is useful because they can gain useful genes = useful characteristics = advantage over competitors = survive = reproduce = pass on allels
    • Plasmids may contain
      • Genes for AntiB resistance = genes for enzymes that help break down AntiB Genes that help Microrgs invade host = genes for enzymes that break down host tissue  Genes that mean microorgs can take up different nutrients = enzymes that break down sugar not normally used by widely accsessiable

11. M)Other Genetic Markers in Plasmids

    • help identify which bacteria have taken up plasmid (other than antib resistnace) Antib resistance avoided as can encourage bacteria and harm human kind 
    • Fluorescense
      • Inserted as a marker gene (or genetic markers) along with the desired gene  = bacteria that take up recomb plasmid will also take up the marker gene codes for enzyme that fluoreses in UV light

12. N)Human Insulin

    • People with type 1 diabeties need to inject insulin to regulate blood glucose concentrations  genetically engineered insulin is identical to human insulin so is better then insulin from animals 
    • Made artifically
      • 1. Mrna located is extracted from B cells in islets of langerhan 2. Mrna with code for human insulin identified and isolated 3.incubate with Reverse transcriptase (retro virus that makes single straned DNA from RNA), nucleotides, dna polymerase to make single DNA made into double DNA = the gene for insulin 4.Add lengths of single standed guamine to the ends (add single stranded cyto to ends of plasmid) 5. K) takes place 6. The transformed bacteria containg the insulin can now produce insulin they are now grown on large scale in fermenter and insulin can be extracted 
    • Made Using K) method
    • Ecoli
      • Bacteria that can take up insulin gene and produce it 
    • 2 Exam Qs
      • (4)Describe how isolated human insulin gene is insterted in bacteria plasmid Using RE  Sticky ends on plasmid that are complementary H bonds a-t c-g Ligase seals nicks in sugar phosphate back bone 
      • People who have one form of diabetes are unable to make insulin. In order to control blood sugar concentration, these people need to receive insulin. Originally, insulin was obtained from animals, such as pigs. Now, bacteria are transformed by genetic engineering to make proteins, such as insulin. This is the source of the majority of insulin now used by diabetics. Describe how genetic engineering has been used to produce human insulin and the advantages of obtaining insulin in this way. [8]    
      • 1 identify / find, gene (for insulin) / length of DNA coding for insulin; 2 obtain / isolate / extract, gene / length of DNA (for insulin); obtain / isolate / extract, mRNA (for insulin); 3 restriction enzyme / named e.g.; reverse transcriptase 4 cut plasmid; cut plasmid;5 use same restriction enzyme; use restriction enzyme / named e.g.;6 ref to, complementary ends / sticky ends / described;7 insert, gene / AW, into plasmid;8 recombinant DNA;9 plasmid uptake by bacteria;10 identify those bacteria that have taken up the plasmid;11 provide with, raw materials / nutrients;12 fermenter / bioreactor;13 bacteria produce insulin;14 extract and purify / downstream processing;15 AVP; e.g.. detail of uptake by bacteria method of identifying those that took up plasmid PCR ligase 7 max16 advantage 1; e.g. more reliable supply17 advantage 2; greater / faster, production overcomes ethical problem described less risk of disease less risk of, rejection / side effects human insulin so more effective 8 maxQWC – clear, well organised using specialist terms; award QWC mark if four of the following are used 1gene plasmid restriction enzyme complementary named e.g. of a restriction enzyme sticky end reverse transcriptase recombinant DNA fermenter / bioreactor [9]   

13. O) Golden Rice

    • Is a genetically engineered plant  it is genetically engineered to include a gene from a daffodil 
    • Why is G rice necessary?
      • Golden rice is needed is eastern asian coutrys that have a rice diet Rice lacks Vitamin A (also called retinol) & B carotene which makes the precursor  Vitamin A is needed to proudce Rhodospin visual pigment in the eye, A lack of rhodospin can lead to night blindness reduce resistance to bacterial and viral infections caused by microogs
    • Making Golden Rice
      • isolate gene from daffodils (makes b carotene) use the K spec point  process Inserted into bacteria that naturally infects plants Bacteria mixed with rice embryos golden rice would express gene for b carotene  Grown on a selective medium so only G rice grow 
    • Where do we get our Vit A
      • From meat like liver we can make retinol from the precursor b carotene 
    • Problems with G Rice
      • small amounts of B carotene in rice so therefore needed to eat huge amount for normal daily intake of B carotene  Just a PR stunt 
    • exam q
      • potential benefits; reduce vitamin a defincy for people in asia, reduce eye problems - night blindness 
      • possilble ethical concerns; reduce rice genetic variation, clone may suffer from one disease, hydrilisation with wild rice, rice may not grow in all areas where needed, doubts as to whether vit a content is effeicent 
    • exam q
      • explain what is meant by genetically engineered - possess foreign/new gene 

14. P) XenoTransplant

    • the transfer of cells, tissues or organs from one species to another
    • Why is it necessary?
      • Cure for organ failure  Donor list if increasing  Many people die waiting for organ Xeno can reduce wait and deaths Not enough Donours Certain pigs have organs very similar to ours
    • How?
      • Need to be close match between donor and recepiant to avoid immune system attack - occurs when human antibodies attach the sugars in PM of the pig organ cells, sugar is made by GGTA1  - if GGTA! is not there then there is no immune response - researchers have located gene that codes for enzyme GGTA1 & have genetically engineered pigs without the gene or with a silence gene  they do this by removing enzyme the nucleas is transferred into an unfertilised animal egg cell (nuclear transfer) the  egg cell is stimulated to divide into an embryo and produces no cell surface protein
      • Or gene for human cell surface proteing are inserted into the animal dna injected into newly fertalised embryo  genes intergrate into animals dna  then produce human cell surface proteins = no rejection 
      • GGAT1
    • Problemooos
      • ETHICS: Is it right? will the GM pig be less healthy/willi it suffer Pigs can be regarded as unclean animals in some religons  PROBLEMS:will huge number of immunosepressent drugs be needed which could put patient at risk of other infections 
    • 2 Exam Qs
      • Define the term antigen
      • stimulates an immune response  produces on or more antibodies
      • suggest why proteins presented on the cell surface are able to stimulate an immune response more effeictvley than proteins dissolved or suspended in the blood or tissue fluid
      • stimulate cell = immediate immunity antigen in body longer antigen in blood only stimulate hormonal immune system

15. Q) Gene Therapy

    • Genetic disorders are disorders that are inherited and are caused by abnormal genes or chromos  gene therapy could be used to cure these disorders  involves altering allels/genes inside cells  - if cuased vy 2 recessive adda a working dominant  - if caused by a dom you can silence it by sticking a piece of dna in the middle  Insert into cell using Spec point K)
    • Q) different Types of Gene Therapy
      • Somatic
        • adding and killing genes in body cells - particularly cells most affected by disorder, not sex cells. Not passed on to next generation and often needs top up as one cells have divided the gene is no longer present  Kill genes like cancer  add parts of gene that is not there due to mutation
        • Exam Q
          • A couple who already had a child affected by Cystic fibrosis (CF) underwent pre-implantation genetic diagnosis. In this process, a single cell, taken from one of several three-day-old embryos created by in vitro fertilisation (IVF), can be tested for CF. The resulting DNA banding pattern produced by electrophoresis is shown in the figure below.(a) Using the information above, *child has allel 2 that mother, child and father have, not allel 1 that hasn't moved as far in the electropherises gel* (i) explain why the three-day-old embryo will develop CF; Go to page 2 for asnwer   
          • two recessive alleles/homozygous recessive/two of allele 2; no, normal dominant/allele 1; homozygous same allele as affected child; REMEBER MOST GENETIC DISORDERS ARE PRODUCED BY RECESSIVE ALLEL    
        • exam q
          • potential benfits; cure/reduce symptoms/better quality of life/less medication treat cystic fibrosis, scid, parkinsons, lca, extended life span and save lives potential ethical concerns; virus vector may cause disease, procedure may be invasive, temporay, immune system could reject, animal testing concens.
      • Germ Line
        • Sex cells . Changing genes/allels in cells that go on to from gametes = zygotes ALL OF CELLS of offspring carry this modification  no need to top up  and inherited  can lead to designer babys
      • Advantages
        • Prolong lives of people with G disorders Better Qual of Life  Carriers could concieve babies that don't have disorder Decrease number of people with G disorders, early treatment, 
      • Disadv
        • Short lived effects (somatic) Multiple Treatment (som) Difficult to get allel into specific cells Immune response on vectors Allele inserted in wrong place = cancer New allel could be over expressed producing to much of missing protein Disorders caused by multiple genes hard to treat
      • 2 Exam Qs
        • discuss advanatages and disadvantages of genetic screening
        • (c) advantage 1 can identify presence of disorder ;2 removes uncertainty ;3 allows early treatment ;4 which may improve, life expectancy / quality of life ; A avoid unncessary suffering5 allows, informed choice about having children / planning healthy family ;6 allows IVF and, embryo screening / preimplantation genetic diagnosis (PGD) ;7 allows fetal testing and termination ;8 choice, re donation / adoption ;9 AVP ; e.g. detail of donation: AI(D) / egg donation / embryo donationmaximum 5 on advantages disadvantages 10 false, positives / negatives ;11 may not be test for all mutations ;12 only small number tests available / not available for all conditions ;13 simple presence may not result in condition ;14 confirmed presence gives stress / fear ;15 problem re, telling / testing, rest of family ;16 discrimination by, employers / insurers ;17 ethics of termination ;18 AVP ; e.g. detail of problem of test, risk of test procedure,diagnosis and elimination rather than treatment, increasein, intolerance / discrimination, of disabled, ‘designer’problem maximum 5 on disadvantages8 maxQWC – clear well organised using specialist terms; 1must include both advantages and disadvantages and two termssuch aslife expectancy, quality of life,IVF, PGD, PGH, AI(D),amniocentesis,CVS, karyotype,false positive, false negative   
        • explain why researches are more worried about dna vaccines then protein vacciens 
        • disrupt genes  taken up by bacteria  super bug irreversible not know long term effects 

16. Ethical Issues

    • Animals
      • suffering religion 
    • Plants
      • Damage enviro Harm health  To other Orgs Safe to eat? Long term affects not yet identified 
    • Microorgs
      • none