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12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe asexual reproduction Involves only one organism Individuals produced are genetically identical i.e. clones Give examples of natural and artificial cloning Natural: bacteria; plants from suckers, blubs, corms Brought about in plants by cell culture What is micropropagation Cloning plants Growth of a plant from part of a plant or a few cells in suitable growth media Describe the steps involved in plant tissue culture Explants placed in sterile, aerated medium Cells divide by mitosis to form callus; callus is subdivided Each piece differentiates into a plantlet Plantlets transplanted into sterile soil when grown to suitable size Define clone A group of genetically identical organisms formed from a single parent as a result of asexual reproduction or by artificial means 1/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Define in vitro fertilisation What does embryo cloning in animals involve A technique which involves mixing the egg or oocyte with sperm in a dish where fertilisation takes place IVF in a petri dish of the best sperm and eggs Splitting of an embryo before cell division And the subsequent growth of cells into many individual embryos What is meant by the term stem cell An undifferentiated cell capable of dividing to give rise to cells which can develop into different types of specialised cells Describe cloning by nuclear transfer • Cells taken from tissues of udder of donor sheep; cultured in medium that prevents division • Unfertilised egg removed from recipient sheep and nucleus is removed • Donor and recipient cells fused together by a gentle electric pulse, allowed to divide to 8cell stage - ball of cells • Developing embryo implanted into the uterus of another sheep - surrogate; lamb born identical to donor sheep Describe the advantages of cloning Speed of production, production of large quantities, identical genetic line of organisms Enables desirable qualities to be preserved for the next generation (by conventional reproduction beneficial mutation would be lost due to reshuffling) 2/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the disadvantages of cloning Describe how cloning is used to conserve rare breeds In mammals, very expensive and unreliable Inadvertent selection of disadvantageous alleles Offspring may show long-term or unforseen effects e.g. premature ageing In plants - disease/entry of pathogens may cause problems Embryos of young animals are bisected and successfully transplanted into a surrogate mother of a common breed to produce a new individual of a rare type Describe what tissue engineering involves? What are its applications Inducing living cells to grow on a framework of synthetic material to produce a tissue e.g. skin Treatment of extensive deep burns; blood vessel replacement, bone and cartilage repair and treatment of degenerative nerve diseases What are the implications of cloned stem cells in terms of organ transplantation Prevent immune rejection Reduce problem of organ shortages Describe the technique to produce a cloned organ Mature cell taken fro patient and nucleus removed Nucleus removed from human ovum; mature cell nucleus transferred into empty ovum Ovum divides into a ball of stem cells Stem cells isolated and cultured with appropriate growth factors allowing their growth into the desired organ 3/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Outline the ethics concerning the use of stem cells • Come from surplus embryos not placed in uterus; embryos destroyed once stem cells removed; some consider this unacceptable even though embryos wouldn't develop anyways • Health benefits inc parkin, alz, heart, liver outweigh • Other sources of stem cells e.g. bone marrow have more restricted medical applications • Slippery slope to reproductive cloning, devalue human life - illegal in UK Define totipotent Cell which can differentiate into a body cell Define meristem Growing points where cells divide rapidly by mitosis Why can micropropagation be used as a cloning technique Name some conventional methods of plan propagation Many plant tissues are totipotent Grafting Taking cuttings 4/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Which tissues in plants aren't totipotent Xylem and pholoem Describe some advantages of micropropagation • Large numbers of plants grown in controlled conditions ensuring greater survival rate •Less time consuming as pollination, seed production, seasonal constraints aren't required •Good quality stock selected - e.g. disease resistance •Crop uniform - commercial •Large plant/area ratio - heating and lighting •Genotypes preserved; reduced space for transport Describe some applications of cell cultures for medical and research purposes The culture of viruses for vaccine production Production of monoclonal antibodies Cell replacement therapy, tissue engineering Describe some disadvantages of micropropagation Sterile conditions must be maintained Plants genetically unstable increased mutation rate; regular inspection required to remove defects Describe the aims of the Human Genome Project Identify all genes in human DNA Determine sequences of 3 billion bp Store info in databases Improve tools for data analysis Transfer related technologies to private sector Address ethical, legal, social issues that may arise 5/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics What is meant by the human genome? All the DNA sequences contained in the chromosomes of an organism What is meant by the term gene probe? Short piece of DNA, whose sequence is complementary to mutated sequences What are the implications of the Human Genome Project in terms of inherited disease Find exact base sequence changes that cause the disease Describe how genetic screening is carried out DNA obtained from patient and scanned for mutated sequences Gene probes used to seek their complement among the 3 billion base pairs of individual's genome Mutated sequence present = binding and resultant flagging of mutation Describe another method of DNA testing other than gene screening Patient's DNA sequence of gene is compared with normal gene Expensive 6/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Outline the main uses of genetic testing Describe the use of genetic testing in terms of cystic fibrosis • Carrier screening (recessive) •Pre-implantation diagnostic testing •New-born baby screening •Pre-symptomatic testing for predicting adultonset disorders - Huntington's •Pre-symptomatic testing for estimating risk of developing adult-cancers + alz •Confirmation individual has disease •Forensic/identity testing Determine whether person is carrying faulty gene Carrier parents may decide not to have children Describe the use of genetic screening in terms of cancers and alzheimers Determines likelihood of developing diseases during adulthood Health authorities can target early diagnosis, advice on reducing risks Describe the advantages of gene testing Improves lives: diagnosis + treatment; children with diseases; identify high risk Describe some problems concerning commercialised gene tests Targeted at pre-symptomatic people with high risk Difficulty interpreting positive result because may be interactions between several genes/environment 7/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe some limitations of gene testing What is meant by gene therapy? Lab errors misidentification/contamination Uncertainties in interpretation risk of stigmatisation, anxiety, discrimination etc Social concerns: who has access to genetic info, how is used? Who owns/controls info; should parents have right to test children for adult-onset diseases; Danger of one day producing human clones A technique whereby a defective gene is replaced with a gene cloned from a healthy individual to provide a potential cure for a genetic disorder What is the main obstacle concerning gene therapy? Developing gene delivery system Inserting normal versions of gene into person's cells Ensuring their correct function What are the two ways of replacing defective genes Gene therapy involving somatic cells - therapeutic but not inherited Germ-line therapy - introduction of corrective genes into germ-line cells What is meant by the term liposome? Minute spheres of lipid molecules, capable of carrying DNA inside them 8/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describes the problems that arise due to cystic fibrosis Produce thick, sticky mucus from epithelial cells lining certain passageways in body Pancreatic duct becomes blocked preventing enzymes reaching duodenum Bronchioles and alveoli of lungs become clogged, causing congestion and difficulty breathing. Mucus difficult to remove - recurrent infections Describe why cystic fibrosis occurs, with reference to its deficient gene Cystic fibrosis trans-membrane regulator (CFTR) transports Clions out of cels into mucus Sodium ions flow out of cells, and water passes out by osmosis So mucus is of a watery consistency Mutation means protein is one aa different thus cannot perform funtion Describe the use of liposomes in treating cystic fibrosis Gene therapy Genes inserted into liposomes can pass through lung epithelial cells Aerosol inhaler delivers the liposomes Liposome and cell surface membrane fuse, DNA released inside cell Gene expressed Describe the use of viruses in treating cystic fibrosis Virus rendered harmless Virus cultured in epithelial cells along with plasmids with normal CFTR gene Gene incorporated in viral DNA Virus isolated and introduced to patient by inhaler Virus injects DNA which includes normal CFTR gene into epithelial cells of lung For what can the current gene therapy treatments for cystic fibrosis not fix? Cannot solve digestive problems 9/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics 10/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the formation of a liposome How effective is gene therapy? How is cystic fibrosis treated on a day-to-day basis? Plasmid DNA containing theraputic gene incubated with empty liposomes -ve DNA binds with +ve liposomes and plasmids absorbed Liposomes containing plasmid DNA are called lipoplexes Short lived - usually repeated in 30-day intervals May be immune response in patient But advantages outweigh the caveats Frequent daily chest physiotherapy massage to keep airways open What is meant by genetic counselling? Advice for people with a family history of a genetic disease - for whether to have a child Closely linked with genetic screening - informs advice Advice based on: history of disorder in family; whether parents closely related; frequency of faulty gene in population Allows parents to make personal decisions Describe different methods of genetic screening Blood tests - e.g. for CF Amniocentesis - withdrawing amniotic fluid during early stages of preg. Cells that have floated away from surface of embryo. Analysed microscopically Chorionic villus sampling - tiny samples of foetal tissue withdrawn from uterus and cells cultured and examined under microscope 11/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Outline the advantages and disadvantages of gene therapy D: invasion of privacy; increased number of abortions; high risk group for insurance purposes (expensive/impossible to obtain); decisions as to whether to have a child; use of gene therapy for wrong reasons - improving appearance of child What is meant by the term clone A population of genetically identical cells or organisms What is meant by the term restriction enzyme? Enzymes that cut DNA molecules between specific base sequences What is meant by ' sticky ends'? The two ends of the 'foreign' DNA segment. They have a short row of unpaired bases that match the complementary bases at the two ends of the opened-up plasmid DNA sample Describe the uses of genetic engineering Genes into bacteria to make useful products e.g. insulin Genes into plants and animals to acquire new characteristics e.g. resistance to disease Genes into humans so no longer suffer disease like CF 12/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Outline the key steps in genetic engineering Isolation of DNA fragment Insertion into vector Transfer of DNA into suitable host cells Identification of host cells that have taken up gene by use of gene markers Cloning of host cells Describe how, in genetic engineering, DNA is identified and isolated Gene probe with complementary sequence finds gene on DNA. Restriction endonucleases cut DNA into small pieces allowing genes to be isolated - sticky ends Or reverse transcriptase: mRNA for gene extracted from cell; enzyme added (from retroviruses) makes copy DNA (cDNA) from mRNA DNA Polymerase added to form dsDNA What is EchoR1 What is the advantage of using the reverse transcriptase method? What are the advantages of using genetic engineering to synthesise insulin? Restriction enzyme Palindrome 3'GAATTC5' and 5'CTTAAG3' Cuts between Gs forming sticky ends Using mRNA carries specific code for gene and not introns found in DNA Not used from animals Large quantities produced quickly and p cheaply 13/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics 14/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics What is meant by the term plasmid? Circular loop of DNA found in bacteria. Known as a vector What is the function of DNA ligase? Ligation (sticking together) of 2 portions of DNA What is meant by recombinant DNA? DNA which results from the combination of DNA fragments from two organisms Describe how a gene is inserted into a vector in genetic engineering Why is the same restriction enzyme used to cut donor and vector DNA Plasmids separated from cell debris after bacterial cell wall is dissolved Rest. endo. used to cut plasmid leaving sticky ends Donor and vector DNA mixed together - sticky ends bind; stuck together by DNA ligases Recomb. plasmid taken up by treated bacteria, which multiply in a fermenter To ensure the sticky ends are complementary 15/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the transfer of DNA into the host cell, including the use of genetic markers What is meant by the term transgenic? Plasmid used contains gene for antibiotic resistance Cells that take up recombinant plasmid also take up this gene Bacteria cultured in ampicillin; only bacteria containing this plasmid will survive (though some plasmids may not contain the gene Gene replicated every time cell divides An organism that has had its genotype altered producing a new strain of organism. Also known as genetically modified organisms Describe how bacteria are used to genetically modify crops Certain bacteria can cause plant cells to multiply and form tumours by inserting plasmid DNA into genome of plant Rest. enz. used to remove tumour-forming gene, replacing it with a gene for e.g. drug resistance using methods outlined before Thus when recombinant bacteria infect plant, gene for drug resistance added to genome Describe two examples of GM crops Soya: major source of food; treated to be tolerant to weed killer; sprayed onto crops but only weeds die; this breaks down in soil into harmless component Tomatoes: ripen when enzyme breaks down pectin in cell wall; complement of gene for enzyme added to genome. Thus both enzyme and complement transcribed forming two mRNAs which form a ds-mRNA molecule, which cannot be translated - Flavr Savr; longer shelf life of tomatoes Describe the reasons why some are opposed to GM • Pollination - genes transfered to plants/wild counterparts with unforeseen effects • Some contain marker genes for antibio resist - concerns of transfer back to bacteria of intestine of consumer 16/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics some are opposed to GM crops of intestine of consumer • Commercialisation leads to decrease in biodiversity of crops meaning susceptibility to attack increases • Commercial growth could comprimise organic farming due to cross pollination by wind and insects 17/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the benefits of the use of GM crops What is meant by the term electrophoresis? What facet of human DNA means genetic fingerprinting can be carried out Describe how genetic fingerprinting is carried out Describe the principal by which gel electrophoresis is governed Improved food: flavour + keeping qualities Introduction of nitrogen-fixing genes reducing dependence on artificial fertilisers Resistance to insects, weeds and diseases Resilience to disease in animals esp in 3rd world Exposing fragments of DNA to an electric current in a gel trough Much of DNA has no known function Non-coding DNA sequences vary in length - HVRs and STRs The lengths of these regions (alleles) are passed onto offspring • DNA extracted and cut into small frags by rest. endo.• Fragments separated by electrophoresis• Southern blotting - trough covered in nylon membrane and fragments transferred• Radioactive DNA probes attach to specific parts of the fragments and any unbound fragments wash off• Membrane placed under X-ray film and image develops• Autoradiograph reveals pattern of light and dark bands (dark = presence of radioactive DNA) - unique DNA is negatively charged so will be repelled by negative charge and attracted to positive charge Larger DNA fragments will encounter more resistance (hydrogen bonds etc) so will move more slowly Thus in a given time a larger DNA fragment will have moved less far than a shorter DNA fragment Hence DNA fragments can be separated by length 18/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics What are used in place of radioactive probes in modern genetic fingerprinting How is a paternity test carried out Chemiluminescent probes Fingerprint of WBCs of mother and father Bands from mother subtracted from child's patten If father - must possess all remaining bands What is meant by the term primer? Short sequences of nucleotides What is in the reaction mixture for PCR? Sample of DNA Buffer DNA polymerase Nucleotides Primers What is the function of primers in PCR? Act as signals to the DNA polymerase to start copying 19/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the process of PCR Why to forensic scientists often use PCR when producing a genetic fingerprint? Target DNA heated to 95ºC - 2 single strands Solution cooled to 55ºC triggering primers to joint to complementary base sequences on each of the single strands of DNA. This triggers DNA replication Solution heated to 70ºC and DNA polymerase catalyses synthesis of complementary strand for each of the single strands of DNA; Repeat Increasing the quantity of DNA because the sample obtained at crime scene may be very small Describe some issues of privacy that arise in the study of genetics Who has access to data Insurance companies Describe some advantages of genetic engineering Large-scale production of complex proteins and peptides that cannot be made by other methods Production of higher yielding crops, drug. res. keeping etc Health benefits for treating genetic diseases Outline some concerns with genetic engineering Germ-line therapy - insufficient knowledge on the interaction between genes - unforeseen effects in future generations Impossible to predict consequences of genetically engineered animals/plants released into environment 20/21 12/10/2015 StudyBlue Flashcard Printing of 5.6 Applications of Reproduction and Genetics Describe the potential hazards of genetic engineering Change in gene function microorganism into pathogen Bacteria may result in incorporation of recombinant DNA into other species e.g. herbicides into weeds Transfer of antibiotic resistance genes into pathogens Tampering with oncogenes, triggering cancer Describe some problems associated with recombinant DNA technology • Technically complicated - expensive on industrial scale • Identifying genes of value on huge genome • Synthesis of required protein may require several genes to code for several polypeptides • Treatment of human DNA with rest. enz. produces millions of fragments of no use • Not all eukaryotic genes can express themselves in prokaryotic cells What are exons? Regions of DNA that code for proteins 21/21