Genomes and gene technology

Genomes and gene technologiesBy Daniella Di-FonzoCandidates should be able to: (a) outline the steps involved in sequencing the genome of an organism; (b) outline how gene sequencing allows for genome-wide comparisons between individuals and between species (HSW7b); (c) define the term recombinant DNA; (d) explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product (HSW6a); (e) describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes; (f) outline how DNA fragments can be separated by size using electrophoresis (HSW3); (g) describe how DNA probes can be used to identify fragments containing specific sequences; (h) outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments; (i) explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase;

Specification(j) state other vectors into which fragments of DNA may be incorporated; (k) explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product; (l) describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment; (m) outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid; (n) outline the process involved in the genetic engineering of bacteria to produce human insulin; (o) outline the process involved in the genetic engineering of Golden RiceTM (HSW6a); (p) outline how animals can be genetically engineered for xenotransplantation (HSW6a, 6b); (q) explain the term gene therapy;(r) explain the differences between somatic cell gene therapy and germ line cell gene therapy; (s) discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms (HSW4, 6a, 6b, 7c).

Sequencing a genomeGenome can mean all genes of an individual organism or all genes in a population of organisms.Gene technologies used to study genes and their functions include: Polymerase chain reaction (PCR) Cutting out DNA fragments using restriction enzymes Gel electrophoresis DNA probes

Sequencing a GenomeIf a whole genome needs sequencing: use PCR to make multiple copies of all the DNA. Squeeze through a tiny hole under high pressure. Cuts DNA into 2000- 10000 base pairs. These can be shortened to make it easier to sequence them.Make multiple labelled copies of these lengths of DNA. They are mixed with a primer (up to 20 base pairs of DNA, complimentary to the start of the DNA fragment you want), free nucleotides, DNA polymerase (to create new DNA strands) and nucleotides labelled with fluorescent dye. Each base has its own colour and each labelled nucleotide with stop the chain from growing.The DNA polymerase attaches free nucleotides to the lengths of DNA. Most of the copies wont be complete due to the labelled nucleotides. This means lots of different length chains.This is then separated using electrophoresis so the DNA is drawn towards the positive end of a capillary tube.A computer records the colours as they pass, the shorter the chain, the faster it travels, so if there are enough fragments, the computer will be able to work out the sequence of bases in that particular length of DNA.This is then done for every piece of DNA and then a computer program works out the overall sequence of nucleotides in the whole genome.

PCRDNA is denatured by heating to 95C, to break hydrogen bonds of the double helix of DNA, so the base pairs are exposed.The mixture is then cooled to between 50C - 65C so the primer (up to 20 pieces of DNA complimentary to the start of the DNA fragment you want) can bind (anneal) to the strands.The mixture is then heated to 72C so DNA polymerase can DNA strand.PCR can be used to make millions of copies of a DNA fragment in just a few hours.The DNA polymerase lines free nucleotides alongside each template strand, base pairing means new complementary strands are formed.Two new copies of each DNA strand are formed from one cycle of PCR.The cycle starts again with the mixture being heated to 95C and all four strands are used as templates.Each PCR cycle doubles the amount of DNA from the previous cycle.

ElectrophoresisThis separates different DNA fragments according to their lengths.A tank containing pure agar (agarose gel) is set up.A direct current is passed through the gel and the DNA fragments (which carry a small negative charge) are attracted to the anode (positive electrode)The smaller the fragment, the faster they move.Labelled nucleotides allow us to use time to sort longer DNA strands from shorter ones.Electrophoresis (cont.)Used for DNA profiling.Phosphurus 32 is the radioactive isotopeWe can also use distance to sort the strands.When the current is turned off, the DNA fragments have ended up in different places and arent immediately visible.Transfer them onto absorbent paper placed on top of the gel and heat so the two DNA strands in each molecule separate from each other.Short sequences of single stranded DNA are added, these are labelled using fluorescents or radioactive isotopes.These pair up with the DNA fragments on the paper and can be detected using UV light or X-ray film.Then we can match the marks to the positions of DNA fragments on the agarose gel.Probes form hydrogen bonds with stretches of DNA close to its complimentary sequence

Restriction enzymesCut DNA backbonesRestriction nucleoases that cut DNA at specific points.Each particular enzyme cuts DNA at a different point- (the restriction site)Less than 10 bases long.Catalyse a hydrolysis reaction that breaks the phosphate sugar backbone of the double helix. Leaves exposed bases called sticky ends.You can get a DNA fragment from a length of DNA using restriction enzymes.DNA ligaseCatalyses condensation reaction that joins the phosphate- sugar backbones in the helix together.Both pieces of DNA need to have been cut with the same restriction enzyme, so the sticky ends are complimentary and can hydrogen bond together. DNA ligase then seals the backbone.The resulting DNA is recombinant DNA.Genetic engineeringUsing technology to change genetic material of an organism.DNA containing lengths from two different species is called recombinant DNAOrganisms with altered DNA are transformed organismGenes can be manufactured instead of extractedAn organism with DNA from another organism is a transgenic organismEthical issues- some people believe.using antibiotic resistant genes as markers may increase number of antibiotic resistant pathogens in our environmentMay encourage farmers to monoculture and decrease biodiversity and leave the whole crop susceptible to disease because they are genetically identicalEngineering animals for xenotransplantation may cause sufferingMay produce herbicide resistant weedsCompanies may exploit farmers in poor countries by selling them crops they cant affordSome people worry humans will be genetically engineered, e.g., to be smarterHuman insulinPeople with type 1 diabetes need to inject insulin to regulate their blood glucose concentrationUsed to be extracted from pigs, now its mad by genetically engineered bacteriaGene for human insulin is identified and isolated using restriction enzymesA plasmid is cut open using the same restriction enzyme used to isolate the geneThe insulin gene is inserted into the plasmid to make recombinant DNAThe plasmid is taken up by bacteria and any transformed bacteria are identified using marker genesThe bacteria are grown in a fermenter and human insulin is produced as bacteria grow and divideThe human insulin is extracted and purifiedAdvantagesMore effective than animal insulin and less chance of rejection because its identical to human insulinCheaper and faster to produce, more reliable and larger supply of insulinOvercomes ethical or religious issues arising from using animal insulin

Golden rice Genetically engineered to produce beta carotene which is used to produce vitamin A. Being used to reduce vitamin A deficiency in places like south Asia and parts of AfricaPsy gene is extracted from daffodil and crtl gene extracted from soil bacterium using restriction enzymesA plasmid is removed from Agrobacterium tumefaciens and cut open using the same restriction enzymesThe genes and a marker gene are inserted into the plasmidThe recombinant plasmid is put back into the bacteriumRice plants are incubated with the transformed bacteria which infect the plant cells Rice plants are grown on a selective medium so only the transformed plants will be able to grow because they contain the marker needed to grow on this mediumxenotransplantationXenotransplantation is the transplantation of cells, tissues or organs from one species to anotherAnimals may be used as organ donors for humans when there is a shortage of human donorsHowever, there is a risk of rejectionScientists are trying to eliminate this risk by:Inserting genes for human cell surface proteins into a newly fertilised animal embryo. The genes integrate into the animal DNA lowering the risk of rejectionGenes for animal cell surface proteins are inactivated. Removed from the nucleus of animal cell and then transferred into an unfertilised animal egg. The egg cell is then stimulated to divide and the animal created doesnt produce animal cell surface proteinse.g., a pig that doesnt have the enzymes needed for it to make a sugar that humans dont have but pigs usually do.Gene therapyGene therapy alters alleles inside cells to cure genetic disorders. If its caused by two recessive alleles, then a dominant allele can be added to make up for them. If its caused by a dominant allele, this can be altered by putting a bit of DNA in the middle of it.The allele is inserted into cells using a vectorViruses, plasmids or liposomes are different vectorsTwo types of gene therapySomatic alters the alleles in body cells. Offspring can still inherit the disease e.g., cystic fibrosisGerm line- alters the alleles in sex cells. Offspring cant inherit the disease. Currently illegal in humans AdvantagesdisadvantagesProlong lives of people with genetic disordersEffects may only be short lived (somatic)Give better quality of life to sufferersPatient may need multiple treatments (somatic)Allow them to have offspring with out disorder (germ line)Might be difficult to get allele into specific body cellsDecrease number of sufferers (germ line)May be rejection by immune systemAllele inserted in wrong place could cause problems e.g., cancerInserted allele could be over expressed, producing too much missing proteinDisorders caused by multiple genes difficult to treat this way.There may also be ethical concerns and financial concerns.Dna probesCan identify DNA fragments that contain specific sequences of bases, e.g., see if DNA contains mutated geneTheyre short strands of DNA that have a base sequence complimentary to the one theyre looking forProbes have fluorescent or radioactive labels so it can be detectedCan be used to see if any family members have a gene that causes a genetic disorderA sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresisTransferred to a nylon mb and incubated with probe thats complimentary to mutated geneIf the mutated gene is present, the probe will bind (hybridise) to itMb is then exposed to UV light and if the gene is present, it will fluoresceChain termination- determines order of bases in a section of DNA Single stranded DNA, primer, DNA polymerase, free nucleotides and fluorescently labelled nucleotides are mixed together and put in four tubes. A different modified nucleotide is added to each of the tubes.They undergo PCR, which produces many strands of DNA. Theyre different lengths because they terminate at different points depending on where the modified nucleotide was addedThe DNA fragments are separated by electrophoresis and visualised under fluorescent light The complimentary base sequence can be read from the gel. The smallest nucleotide is at the bottom and by reading the bands from top to bottom, you can build the DNA sequence one base at a timeBacterial artificial chromosomes (bacs)To sequence an entire genome, you need smaller piecesGenome is cut into 100 000 bp using restriction enzymesFragments are inserted into BACs- man made plasmids. Each fragment is inserted into a different BACThe BACs are then inserted into different bacteriaThe bacteria divide into colonies of clones with specific DNA fragments, together they make a complete genomic DNA library DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNAEach piece of DNA is sequenced using chain termination, and the pieces are put back in order to give the full sequence from that BACDNA fragments from all BACs are put back in order to give the entire genome

Comparing genomesComparing genomes of different speciesComparing genomes of the same speciesUnderstand evolutionary relationships. Closely related species will share more DNATrace early human migration. Compare genomes of people in different parts of the worldUnderstand how genes interact during development and how theyre controlledStudy genetics of human diseases to detect particular mutations that lead to an increased risk of a diseaseCarry out medical research on genes found in genomes of other mammals e.g., cancerDevelop medical treatments for particular genotypes