31/ Splicing: removes non functional introns from pre-mRNA, occurs in eukaryotic cells - intervening non-functional introns removed

and exons joined together; Exons- DNA sections coding for proteins and Introns- DNA sections that don't code for proteins; splicing details: Once introns removed, exons rejoined in diff. combinations, hence, single section of DNA (gene) can code for up to 12 diffe rent amino acids - depending on exon order Mutations can affect splicing of pre-mRNA Introns left on mRNA - lead to production of non-functional polypeptides or no polypeptides Role of RNA polymerase (transcription), tRNA and Ribosomes (translation): RNA polymerase - causes bases on template strand to join with individual comp. nucleotides in nucleoplasm, joins together comp. nucleotides to form pre-mRNA and stops joining nucleotides when it reaches stop codon on template strand tRNA - attaches peptide at one end with anticodon on other, transfered to ribosome on mRNA molecule, anticodon on tRNA pairs with comp. codon sequence on mRNA, anticodon on tRNA pairs with comp. codon seq. on mRNA. Further tRNA molecules with peptides attached line mRNA in sequence determined by mRNA basesensuring correct sequence of peptides in polypeptide Ribosomes - controls formation of one polypeptide, attaches to mRNA by small subunit, larger subunit can hold 2 mRNA codons: one [codon] held in P (peptidyl) and other in A (aminoacyl) site - each codon attracts anticodon (comp. base triplet), ribosome moves along mRNA from 5' to 3' end; completion of translation is signalled by 39/ Control of Gene Expression [Cancer control] (p4) Cancer - caused by Acquired Mutations: Prevention - protective clothing, sunscreen and vaccination; Diagnosis - usually after symptoms appear, high risk individuals can be screened for cancer general pop. Not privy to (earlier diagnosis -> increases recovery chances) & if specific mutation known more sensitive tests can be developed (earlier and more accurate diagnosis -> improving recovery chances); Treatment - different for different mutiations, agressiveness of treatment can differ depending on mutaiton (fast-growing cancer- treated with higher doses of radiotherapy or removing large areas of tumor and surrounding tissue), if specific gene known - gene therapy may be able to treat it Cancer - caused by Hereditary Mutations: Prevention - people with family history should avoid gaining extra acquired mutations by avoiding mutagenic agents, if mutation causes high risk of cancer preventative surgery may be carried out (removing organ cancer likely to affect) before cancer develops; Diagnosis screening or increased earlier screening, for family history & those with hereditary mutation can lead to early detection (increasing survival chances); Treatment - different for different mutations, aggressiveness of treatment can differ depending on mutation (fast-growing cancer- treated with higher doses of radiotherapy or removing large areas of tumor and surrounding tissue), if specific gene known - gene therapy may be able to treat it Genetic disorders caused by Hereditary Mutations: Diagnosis - DNA analysis of person with family history of genetic disorder, tested and diagnosed - treatment can begin earlier, knowing if they have or carry disorder - allows determination of likelihood of children having disorder; Treatment - gene therapy, diff. treatment for diff. mutations; Prevention - carriers or sufferers of genetic disorders can undergo preimplantation genetic diagnosis during IVF to prevent offspring having disease (embryos with mutation implanted in womb) 41/ Gene Cloning and Transfer (p1) Fragments of DNA can be produced by: Conversion of mRNA to cDNA (using RT), Cutting DNA at specific palindromic recognition sequences (using RE) and Using the polymerase chain reaction (PCR) to form multiple copies of DNA fragments Process of making protein via DNA technology: Isolation - of DNA fragments with gene for desired protein, Insertion - DNA fragment into Vector (carrier), Transformation - Transfer of DNA into host cell, Identification - of host cells that have taken up gene using gene markers, Growth/ Cloning - of population of host cells. 2 enzymes used to isolate and identify genes from DNA: Reverse transcriptase (RT) - catalyses production of DNA from RNA; process of isolating gene: cells readily producing protein selected, relevant mRNA is extracted from cells, RT used to make DNA from RNA DNA produced known as complimentary DNA (cDNA) - made of nucleotides complimentary to mRNA, to make other DNA strand DNA polymerase builds up comp. nucleotides on cDNA template; double stranded gene is required Restriction endonucleases (RE) - enzymes that cut up DNA; many types of RE cut DNA double-strand at diff. specific 'recognition sequence' of bases, RE can cut between 2 opposite base pairs leaving straight edge (blunt edges). Other RE's cut DNA in staggered fashion, leaving uneven cut in which each DNA strand is exposed unpaired bases or 'sticky ends'; Palindromic sequence - anti-parallel arrangement of bases.'6 bp palindromic sequence' - six anti-parallel base pairs (6 base pairs that read the same in opposite directions) 42/ Gene Cloning and Transfer [In Vitro] (p2) Cloning - using PCR to form multiple copies of DNA fragments; Vitro In (can produce 100bn copies in a few hours) - useful where minute amount of DNA present Extremely rapid : Advantages - only base sequence of DNA needing amplification required Doesn't require living cells (thermostable) - enzyme that joins DNA polymerase - to be copied, DNA fragments the following: PCR requires - short nucleotide sequence with bases comp. to those at one end of each 2 DNA DNA Primers nucleotides together, reaction PCR computer controlled machine, which varies temp. precisely of time period. Thermocycler fragments and : process temp. cycle and - DNA frag.'s, primers & DNA polymerase placed in vessel in thermocycler. Temp. Separation of DNA strands increased to 95oC, causing 2 DNA frag. strands to separate; annealing primers to comp. bases at DNA frag. end , mixture cooled to 55oC Annealing (addition) of Primers (only attach nucleotides to end of existing chain) to copy DNA polymerase for Primers provide starting sequence Primers pre vent 2 separate strands rejoining DNA and - temp. increased to 72oC (optimum temp. for DNA polymerase to work), DNA polymerase DNA Synthesis

 begins (adding comp. nucleotides along each separated DNA strand) at primers on both stands adding nucleotides in sequence until end of chain Both strands copied simultaneously - producing 2 copies of original fragment: once 2 DNA strands completed process is repeated, over 1 mil copies can be made in only 25 temp. cycles and 1 temp. cycle takes 2 mins 44/ Gene Cloning and Transfer [In Vivo 2/3] (p4): Introduction of DNA into host cells (stage 2) - process of incorporation of DNA fragment into plasmid and its reintroduction into bacterial cells; Involves: transformation mixing plasmids and bacterial cells with medium containing Ca ions, Ca ions and temp. change -> increase permeability of bacteria ->allowing plasmids to pass through cell membrane into cytoplasm Not all bacterial cells will posses DNA fragments as: only few bacterial cells take up the plasmid and some plasmids will close up without incorporating the DNA frag. - using bacterial mechanism for resisting antibiotics (produces enzyme that breaks down antibiotic before it Identification can destroy bacterium). some plasmids carry resistance gene for more than one antibiotic - one example is the R-plasmid, Process for Ampicilin: tretracycline and ampiclin carries gene resistance for: All bacterial cells grown on medium containing antibiotic ampicilin bacterial cells that have taken up plasmids will have acquired ampicilin resistance gene these bacterial cells are able to break down ampicilin and survive bacterial cells that haven't taken up plasmid are not resistant to ampicilin and die effective method of showing bacterial cells which have taken up plasmids; but, some cells will have taken up plasmid but closed up without incorporating gene and will have survived - must id and eliminate these cells 45/ Gene Cloning and Transfer [In Vivo 3/3] (p5) (stage 3) Gene Makers - involve using separate gene on plasmid. Second gene easily identifiable as: may be resistant to antibiotic, contain fluorescent protein easily see or produce enzyme whose action is identifiable Antibiotic-resistance gene markers - to identify cells up taking plasmid with new gene replica plating technique is used. Process uses antibiotic-resistance gene in plasmid - gene cut to incorporate required gene (as gene cut - no longer produces enzyme that breaks tetracycline (bacteria that have taken up gene, not resistant to tetracycline) and bacteria identified by growing them on culture containing tetracycline; process is as follows: Bacterial cells that survived first antibiotic (ampicillin)- have taken up a plasmid and are cultured by spreading thinly on nutrient agar plates each separate cell on growth plate will grow into genetically identical colony, sample of each colony transferred onto 2nd replica plate in exact same position as on original plate 2nd plate contains second antibiotic (tetracycline), against which antibiotic -resistance gene will be useless if new gene was taken up colonies killed by antibiotic must be ones that have taken up required gene colonies in exact same positions on original plate are ones that possess required gene. These colonies are made up of GM bacteria and have been transformed Fluorescent gene markers - green fluorescent protein (GFP) - gene to be cloned is transplanted into centre of GFP gene, bacterial cell that takes up plasmid with gene to be cloned will not produce GFP. As bacterial cells with desired gene are not killed, no need for replica plating; result obtained by viewing sample under microscope and retaining bacterial cells 46/ Gene Cloning and Transfer [rDNA 1/2] (p6) - 3 substances produced using GM microorg.'s: GM Microorganisms - produced naturally by bacteria; GM has produced bacteria that increase quantity of antibiotics Antibiotics produced and rate at which they're made - Genetic engineering method (for hormone production) avoids killing animals and need to modify Hormones insulin before its injected into humans; other hormones produced: human growth hormone, cortisone and sex hormones - testosterone and oestrogen - GM (bacteria) manufactured enzymes used in food industry. Including amylases to break down starch Enzymes to produce beer, lipases to improve flavour of cheeses and proteases to tenderise meat - gene inserted comp. to base sequence producing enzyme that Genetically modified tomatoes examples: GM Plants, causes tomatoes softness. mRNA transcribed from inserted gene comp. to mRNA of original - the 2 strands combine to form Herbicide resistant doublestrand, preventing mRNA of original gene being translated - softening enzyme not produced. - when herbicide sprayed on crops, weeds competing are killed, as crops are resistant to herbicide and unaffected. crop - gene added allowing plant to make toxin which kills insects (that eat plant), but harmless to other Disease resistant crops animals (including humans) - 2nd animal made transfer of gene from animal with natural resistance to disease into diff. animal , examples: GM Animals Producing rare and expensive proteins - animals grow larger and at faster rate; addition of fast growing hormones resistant; - via domesticated animals e.g. gene for required proteins inserted alongside gene coding for for use in human medicine [see antithrombin section on next card] proteins in milk, hence protein produced in milk. 47/ Gene Cloning and Transfer [rDNA 2/2] (p7) Use of recombinant DNA tech.: increasing yield of plant& animal crops, improving food nutrient content, introducing resistance to disease and pests, making crop plants tolerant to herbicides, developing tolerance to environmental conditions, making vaccines and producing medicines for treating disease. Use of recombinant DNA to produce organisms that benefit humans:

 Benefits - use of microorganisms (microorg.'s) to produce range of substances, use microorg.'s to control pollution, GM plants can be transformed to produce specific substance in particular plant organ, GM crops engineered to have economic advantages, GM crops help prevent disease, GM animals able to produce drugs cheaply, Gene therapy (GT) used to cure genetic disorders and Genetic fingerprinting used in forensic science Risks - cant predict effect of releasing GM organisms into environment, recombinant gene may pass from one organism to another, DNA manipulation has effect on cells metabolic pathway, GM bacteria may resist gene markers, all genes mutate, long-term consequences of new gene introduction, economic consequences of plants & animals grown in new regions, cost of genetic engineering (justified?) and genetic fingerprinting (reliable forensic tool?) Effect of rDNA on Animals: Anti-thrombin, process of production in goats milk: mature egg removed and fertilised, normal gene for antithrombin production from human added alongside genes coding for milk, genetically transformed egg implanted into female goat, resulting goats cross bred to give heard anti-thrombin containing milk and anti-thrombin extracted from milk - purified and given to humans 48/ 48 of 56 Gene Therapy (p1) - most common genetic disorder in causican population of europe and north america (1 in 20k have Cystic Fibrosis mutation), mutation causes single deletion disease), caused by mutant recessive allele where AAA bases are missing (peptide to be left out of cystic fibrosis trans-membrane-conductance-regulator (CFTR) gene - disabling protein from transporting Cl ions across epithelial membrane - mucus layer builds up (no water diffuses out and carries it away). CFTR is Cl ion protein channel. : mucus congestion in lungs, breathing difficulties, accumulation of thick mucus in pancreatic ducts and Symptoms accumulation of mucus in sperm ducts of Cystic Fibrosis using gene therapy: Treatment - defective gene replaced with a health gene Gene replacement - one or more copies of health gene added alongside defective allele Gene supplementation 2 additional GT techniques based on type of cell being treated: - replacing or supplementing defective gene in a fertilised egg Germ-line GT hence, not passed onto future - targeting affected tissue only (i.e. lungs not sperm or egg, Somatic-cell GT by introducing additional gene; repeated treatment needed as lung cells continually die and are generations replaced. Long-term aim: target undifferentiated SC's that give rise to mature tissues (treatment then affective for life of individual) 50/ 50 of 56 Gene Therapy (p3) using gene therapy: people with disorder don't show cell mediated ) treatment SCID ( Severe combined immunodeficiency immunity and can't produce antibodies - arises when person inherits defect gene for enzyme ADA (enzyme destroys toxins that otherwise kill WBC's); Attempts at treatment disorder with gene therapy: from healthy human and ADA gene inserted into retrovirus RE normal ADA gene isolated using retrovirus growing with host cells in lab to increase number and copies of ADA gene retroviruses mixed with patients T cells (WBC's), retroviruses inject copy of normal ADA into T cells T cells then reintroduced into patients blood to provide genetic code needed to make ADA Treatment effectiveness limited as T cells have 6-12 months lifespan & treatment has to be repeated at intervals; recent treatments transform bone marrow SC's (bone marrow SC's divide to produce T cells), because: effect is short lived (S cells aren't passed somatic-cell GT , limited success to Effectiveness of Gene Therapy to daughter cells - re-treatment necessary), Can induce immune response (gene and vector can induce response, hence often rejected - antibodies often remain to initiate greater response), Using viral vectors to deliver gene presents problem (viruses can lead to toxic, inflammatory immune response), Genes not always expressed (small proportion of introduced genes usually expressed) and Not effective in treating conditions that arise from more than one gene: who decides what is normal and what a disability is? Do disabilities need to be Risks and Benefits of Gene therapy cured or prevented? are disabilities part of genetic variety that makes up all species? Is money for gene therapy better spent elsewhere? what might the long-term consequences of introducing inheritable genes into population be? 51/ Medical Diagnosis (p1) DNA Probes: short, single-stranded section of DNA, with label attached making it easily identifiable; used to identify gene by: probe being comp. to bases on portion of DNA sequence desiring to find, DNA tested for - separated into 2 strands, One separated strand with comp. bases to probe - mixed with and binds to probe, probe binding site identified by radioactive exposure or fluorescence probe emits. Two types: Radioactively labelled probe (nucleotides have isotope - identified via photographic plate - when exposed to radioactivity) and fluorescently labelled probe (fluoresces under certain conditions). DNA Sequencing - Sanger method used to sequence exact order of nucleotides in a section of DNA, using modified nucleotides (terminators) that can attach to next base in sequence when being joined together (4 diff. terminators i.e. A,T,G and C); process of sequencing: First stage - set up 4 test tubes containing: many single-stranded DNA frag.'s to be sequenced; mixture of nucleotides, small quantity of 1 of 4 terminator nucleotides (tt.1 A term., tt.2 T term etc.), primer starts DNA synthesis process and DNA polymerase catalyses DNA synthesis. Binding of nucleotides to template is random process (equal prob. of norm. or term. nucleotide addition), DNA frag.'s in the diff. tt.'s will have varying lengths. Second stage (Gel Electrophoresis) - process: DNA (-ve charge) frag.'s placed on agar gel in petri dish & voltage applied, resistance of gel means larger frag.'s move more slowly, over fixed time period smaller frag.'s move further (experience less resistance), hence - DNA frag.'s separated, sheet of photographic film placed over agar plate for several hours and radioactivity (labelled probe) from each gel frag. exposes film showing where frag.'s situated on gel. Only DNA frag.'s up to 500 bases long can be sequenced in this way. [DNA moves from

55/ Genetic Fingerprinting (p1) Genetic fingerprinting (GF) - technique relies upon fact that genome of an organism contains core (repetitive) sequences of introns (non coding bases of DNA); core sequence number and length varies in every individual (except identical twins). GF takes place in 5 main stages [process]: Extraction - DNA extracted from sample (blood, hair root or semen) Digestion - RE's cut DNA into frag.'s (cut close but not within groups of core sequences) Separation - Frag.'s separated using gel electrophoresis, then, DNA frag.'s transferred from Gel to nylon membrane via Southern Blotting process Hybridisation - radioactive DNA probes added to label the fragments (attach to specific fragments) Development - Membrane with radioactive DNA frag.'s placed on X-ray film; development of X-ray film reveals dark bands where radioactive DNA probes have attached Southern Blotting Process - thin nylon membrane laid over gel, membrane covered with many sheets of absorbent paper (draws up liquid containing DNA by capillary action, transferring DNA frag.'s to nylon membrane in same position that occurred on gel and DNA frag.'s fixed to membrane using UV light Interpreting Result - if there is a match between 2 DNA samples, pattern of bars of each fingerprint visually checked, then passed through automated scanning machine - calculating lengths of DNA frag.'s from bands (obtain data by measuring distances travelled by frag.'s during electrophoresis by standard, known lengths of DNA) and odds calculated of someone having identical fingerprint - closer the match, greater prob. the 2 DNA sets are from same person. 56/ Genetic Fingerprinting (p2) - Used in forensic science to indicate whether individual connected to a crime (from blood Uses of DNA Fingerprinting each or semen at scene), resolve paternity cases (individuals inherit DNA from parents, half father & half mother - hence, DNA GF) and useful in determining genetic variability within corresponding band in parents' on DNA GF should have band population (more closely related 2 individuals closer their resemblance of their GF). Other uses of genetic fingerprinting): GF ( - GF can: using small quantity from crime scene establish whether person was present at crime Forensic science scene; however, close at match between suspects DNA and DNA at crime-scene doesn't mean suspect committed contaminated after crime or belong to close relative , left on innocent occasion crime as, DNA may: have been; probability of DNA being someone else's has to be calculated ) RE (other suspects DNA or chemicals affecting (calculation based on assumption - DNA produces bonding patterns randomly distributed in community). *Like Huntingtons Disease: 3-nucleotide segment at one help diagnose diseases - GF can: Medical diagnosis chromosome end continually repeated, fewer than 30 rep's - unlikely to get disease; 38 + rep's - certain to get disease; 50 + rep's disease occurrence earlier than norm. DNA from person with Huntingtons allele can be cut by ER & DNA GF prepared and matched with various of Huntingtons allele's allowing determination of probability comparing fingerprints found by identify nature of microbial infection and timing of symptom development] and in patients with known pathogens identify, prevent undesirable interbreeding in breeding programmes - GF can: Plant and Animal breeding (individuals with desired gene can be selected for breeding - plants or animals with desirable gene allele determine paternity in animals and establish