Genomes and Gene Technologies Flashcards
(a) Outline the steps involved in sequencing the genome of an organism;
BACs
- Genomes are mapped to identify which part of the genome (which chromosome or section of chromosome) they have come from. Information that is already is known is used; e.g. the location of microsatellites (a repeating sequence of three or four base pairs found in many locations on the genome. Alleles at a locus can have a different number of repetitions of the same sequence).
- Samples are the genome are mechanically sheared into smaller fragments of around 100,000 base pairs long; microsatellites are found to identify which part of the genome the fragment is from.
- The fragments are placed into BACs (Bacterial Artificial Chromosomes; man-made plasmids), and transferred to E.coli cells; bacteria divide, creating colonies of cloned cells (many copies are produced) that contain a specific DNA fragment, which together with the different colonies make a complete genomic DNA library; Clone Libaries.
- Cells containing specific BACs are taken and cultured; DNA is extracted from each colony (from the cells) and restriction enzymes cut them up into smaller (further fragmentation) overlapping pieces/fragments of DNA. The use of different restriction enzymes on a number of samples gives different fragment types.
- The overlapping fragments are then sequenced using the chain-termination method:
Chain termination
Used to determine the order of bases in a section of DNA (gene).
The many copies of the fragments are put in a reaction mixture, of four separate test tubes:
- A single-stranded DNA template (DNA to sequence; fragments above)
- Lots of DNA primer (short, single-stranded pieces of DNA around 10-20 bases long, which anneals to its complimentary sequence in PCR)
- DNA polymerase (the enzyme that joins DNA nucleotides together)
- Free nucleotides (lots of free A, T, C and G nucleotides; dNTPs; DNA nucleoside triphosphate consisting of deoxyribose, a nitrogenous base and three phosphate groups; loss of two phosphate groups supplies the energy for the reaction, forming dNTPs)
- Fluorescently labelled (for detection in automated sequencing machines) dideoxynucleoside triphosphates (ddNTPs); chain-terminating nucleotides that lack a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides; inclusion of a ddNTP terminates a growing DNA chain.
- DNA is denatured to separate its two strands (hydrogen bonds broken); all the BAC fragmentation shit
- Sample is divided into four separate sequencing reactions, containing all four standard dNTPs
- Single-stranded DNA (fragments) is replicated by DNA polymerase using a primer (short length of single stranded DNA complementary to the base sequence at the 3’ end of the chain) to being the synthesis. It forms the starting point from which DNA polymerase can continue replicating the chain; annealing to the 3’ end of the template strand, allowing DNA polymerase to attach free nucleotides.
- A low concentration of one of the ddNTPs (e.g. the C form, ddCTP) is present (a modified nucleotide) so that it is added only rarely to a lengthening chain to terminate it; the DNA polymerase enzyme is thrown off and the reaction stops on that template strand.
- As the reaction proceeds, many molecules of DNA are made. The fragments generally differ in size, as different numbers of nucleotides will have been added; adding the products of all four reactions together produces a set of fragments that end at nucleotides with different bases, differing in length by one nucleotide; a set of nested fragments.
- The nested fragments are then sorted by length by gel electrophoresis, getting the banding by southern blotting.
Automated sequencing can be done by labelling each of the four ddNTPs with a different-coloured fluorescent dye. On separation in a single lane of a sequencing gel, the different colours can be distinguished as the fragments leave the end of the gel and pass through a detector. A computer displays the data as a series of peaks, giving a direct reading of the sequence.
As the strands run through a machine, a laser reads the colour sequence. The sequence of colours, and therefore the sequence of bases can then be displayed
(b) Outline how gene sequencing allows for genome-wide comparisons between individuals and between species;
- The identification of genes for proteins found in all/many living organisms gives clues to the relative importance of such genes to life.
- Comparing the DNA/genes of different species shows evolutionary relationships; the more DNA sequences organisms share, the more closely related they are likely to be. All organisms evolved from shared common ancestors.
- Modelling the effects of changes of DNA can be carried out; e.g. the effects of mutations on genes obtained from yeast that are also found in the human genome.
- Comparing genomes from pathogenic and similar but non-pathogenic organisms can be used to identify the genes/base-pair sequences that are most important in causing the disease itself, thus leading to identification of targets for development more effective drugs and vaccines.
- The DNA of individuals can be analysed; can reveal mutant alleles or the presence of alleles associated with particular diseases e.g. heart disease or cancer.
- Develop medical treatments for particular genotypes; same medicine can be more effective in some patients that others, due to their different genomes. In the future, tailored-medicine may be prevalent sequencing a patient’s genome so that they can receive the most effective medicine for them.
(c) Define the term recombinant DNA;
DNA which has been combined from different organisms into one organism; a section of DNA (often in the form of a plasmid) formed by joining DNA sections from two different sources.
(d) Explain what 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. Involves obtaining and then inserting a gene.
The gene is first obtained by removing it from the donor organism’s genome using restriction enzymes at a specific restriction site. The gene is then inserted into the recipient by a vector, and then inserted into the genome by cutting the genome with the same restriction enzymes used in the removal. The sticky ends of the gene and genome are allowed to anneal and DNA ligase then joins the sugar-phosphate backbone together. This means the gene can be transcribed into a protein, changing the characteristics or capacity of the organism; which is now known as a transgenic organism
- The required gene is obtained
- A copy of the gene is placed in a vector
- The vector carries the gene to the recipient cell
- The recipient expressed the gene through protein synthesis
(e) Describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes;
Also known as restriction endonucleases; enzymes that catalyse a hydrolysis reaction that breaks the sugar-phosphate backbone of DNA at a restriction site, cutting the DNA at two points to form a sticky end. They are derived from bacteria which use it in defence against infection from viruses, such as EcoR1 from E. coli.
- Each restriction enzyme binds to and cuts DNA at a specific target site; a palindromic (symmetrical of both strands) sequence usually less than 10 bases long (4-6 bases normally); the restriction site.
- Different restriction enzymes cut at different specific recognition sequences (palindromic bit), as the shape of the recognition sequence is complementary to an enzyme’s active site.
- Bacterium’s own DNA is protected from attack by not having the active site, or by having it hidden behind chemical markers.
- The two strands of DNA may be cut in the same place, leaving blunt (unstaggered) ends, or cut in different places, leaving sticky ends (staggered) of unpaired bases.
- The DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment out via a hydrolysis reaction, breaking the sugar-phosphate backbone at different places.
- When separate DNA fragments need to be stuck together, DNA ligase catalyses a condensation reaction which joins the phosphate-sugar backbones of the double-helix together; both sources need to be cut with the same restriction enzyme for this to occur, so that the sticky ends are complementary and allows the bases to pair up and hydrogen bond together, sticky ends are annealed, where DNA ligase can then seal the backbone (the phosphodiester/covalent bonds in the sugar-phosphate backbone).
(f) Outline how DNA fragments can be separated by size using electrophoresis;
DNA fragments are separated according to length by agarose gel electrophoresis:
- Fragmented DNA samples are placed into wells at the negative electrode (cathode) end
- The gel is immersed in a tank of buffer solution
- A direct current is passed through the gel for a fixed period of time (usually 2 hours)
- DNA fragments are negatively charged (due to phosphoryl groups); move towards the anode (positive electrode)
- The shorter the fragments, the further they travel in the time allowed
- The DNA fragments are viewed as bands under UV light (fluorescent tag on DNA fragments)/a dye is used that stains DNA molecule
The patterns of bands of DNA are invisible at this stage, unless a blue or fluorescent dye which attaches to the DNA is added. Usually the invisible DNA banding pattern is transferred to a nylon membrane by Southern blotting:
- Gel is covered with a nylon membrane and then by absorbent paper towels
- DNA drawn up onto the nylon membrane by capillary action
- Membrane is heated to denature the DNA to single strands
- A radioactive (32p) DNA probe with a base sequence complementary to part of the wanted sequence is added, the surplus washed off.
- The position of the bound probe can be found by placing X-ray film over the membrane; the emissions from the radioactive probe produce black bands on the developed film.
(g) Describe how DNA probes can be used to identify fragments containing specific sequences;
A DNA probe is a short single-stranded section of DNA that is complementary to the section of DNA being investigated. The probe is labelled in one of two ways so it can be detected:
- Using a radioactive marker so that the location can be revealed by exposure to X-ray film
- Using a fluorescent marker that emits a colour on exposure to UV light
Copies of the probe are added to a sample of DNA fragments and will anneal to any fragment where a complementary base strand is present; thus can used a DNA probe to see if any members of a family have a mutation in a gene that causes a genetic disorder, by identifying DNA fragments that contain specific sequences of bases, locating the target genes on a chromosome.
(h) Outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments;
Polymerase Chain Reaction; A form of artificial DNA replication used in order to amplify small quantities of DNA. It differs from natural replication as it can only replicate small quantities of DNA, primers are needed, and a cycle of heating and cooling is required.
- A reaction mixture is set up contacting the DNA sample, free DNA nucleotides, primers and Taq polymerase (a type of DNA polymerase enzyme; not destroyed by the denaturation step so does not have to be replaced during each cycle)
- DNA mixture is heated to 95oC to break the hydrogen bonds, separating the two strands; denaturation.
- Mixture is cooled to 50-65oC to allow the primers (short lengths of single-stranded DNA complementary to the bases at the start of the fragment you want) to anneal via hydrogen bonding; bind to the strands as the temperature lowers.
- Temperature is raised to 72oC, the optimum temperature for Taq polymerase to elongate the primer; Taq polymerase binds and extends primers using free nucleotides to the unwound DNA via complementary base pairing; new complementary strands are formed.
- When the Taq polymerase reaches the other end of the DNA, a new, double stranded DNA molecule is generated; two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
- The whole process can be repeated many times so the amount of DNA increases exponentially; giving 2n copies of the original DNA.
(i) Explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase;
A plasmid is a small, circular DNA molecule found in prokaryotes. It carries genes that are not essential for cell growth or division, but which confer traits that can be a selective advantage under certain conditions.
The plasmids and the (desired) fragments are both cut with the same restriction enzyme so they have complementary sticky ends; the base pairs anneal and DNA ligase joins together the phosphate sugar backbones (ligation) to form a recombinant plasmid (now codes for the inserted protein).
(j) State other vectors into which fragments of DNA may be incorporated;
A vector is a carrier which carries a piece of DNA from one organism to another; a means of delivering a gene into a cell. It is a carrier into which a DNA fragment containing the wanted gene can be inserted; resulting in recombinant DNA. Vast majority of genetic engineering uses bacterial plasmids as the vector.
Commonly used vectors:
- Liposomes
- Viral DNA e.g. bacteriophages (viruses that infect bacteria)
- Bacterial plasmids
- Hybrid vectors with the propertied of both plasmids and bacteriophages: cosmids, phagemids and phasmids.
(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;
Transformation:
- Large quantities of the plasmid are mixed with bacterial cells, some of which will take up the recombinant plasmid straight away
- Calcium salts are added and ‘heat shock’ is used, the temperature of the culture is lowered to around freezing, then rapidly raised to 40oC, increasing the rate at which plasmids are taken up by the bacteria cells.
- Even so, the process is very inefficient; only 0.25% of bacterial cells take up a plasmid; those that do are known as transformed bacteria; they contain DNA, thus are transgenic (an organism which contains recombinant DNA and can therefore express a gene that is not naturally found in the individual’s genome).
(l) Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment;
- Genetic variation
- Genes that code for resistance to antibiotics; genes for enzymes that break down antibiotics
- Genes that help microorganisms invade hosts; genes for enzymes that break down host tissues
- Genes that mean microorganisms can use different nutrients; e.g. genes for enzymes that break down sugars not normally used.
Bacterial conjugation occurs: The transfer of genetic information in the form of plasmids between bacteria. First, a conjugation tube of plasma membrane is formed between the two bacteria, and then an enzyme makes a nick in the plasmid, allowing one of the strands of the DNA to uncurl and enter the recipient bacteria. As this occurs, the original plasmid replicates, replacing the lost DNA; while the plasmid in the recipient bacterium will do the same. This creates two exact copies of the plasmid in each cell, and the cells moves apart. Since plasmids often carry genes associated with resistance to antibiotics, this swapping of plasmids is of concern as it speeds the spread of antibiotic resistance between bacterial populations.
(m) Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid;
- Not all bacteria take up the (plasmid) vector.
- Some of the bacteria take up a plasmid that has reformed to its original self in the presence of the ligase enzyme; containing no copy of the desired gene.
- Marker genes can be used to identify the ones that have taken up the desired gene.
- Marker genes/genetic markers can be inserted into vectors at the same time as the desired gene; any transformed bacterial cells will contain the desired gene AND the marker gene.
- The bacteria are grown on agar plates and each cell divides and replicates its DNA, creating a colony of cells.
- Transformed cells will produce colonies where all the cells contain the desired gene and the marker gene.
- The marker gene can code for antibiotic resistance; bacteria are grown on agar plates containing the antibiotic, so only cells that have the marker gene will survive and grow.
- The maker gene can code for fluorescence; when the agar plate is placed under a UV light, only transformed cells will fluoresce. The required colonies can now be identified and be grown on a large scale.
(n) Outline the process involved in the genetic engineering of bacteria to produce human insulin;
People with Type 1 diabetes cannot make insulin and need daily injections of the hormone; large quantities can be manufactured using genetically engineered bacteria.
- Preparation of insulin gene; mRNA for human insulin extracted from pancreas cells
- Reverse transcriptase uses mRNA as a template to make single-stranded complementary DNA (cDNA); this is made double-stranded by DNA polymerase.
- A single sequence of nucleotides (GGG) is added to each end of the DNA to make sticky ends
- Preparation of a vector to carry the human gene to a bacterium; plasmid is cut open with a restriction enzyme (same one used to isolate the insulin gene)
- Cut plasmid has a single sequence of nucleotides (CCC) added to each end to make sticky ends
- Plasmid and insulin gene are mixed to that sticky ends form base pairs (forming recombinant DNA).
- DNA ligase links sugar-phosphate backbones of plasmid and insulin gene.
- Formation of genetically engineered bacteria; plasmids are mixed with bacteria in the presence of calcium ions to increase porosity.
- Bacteria take up plasmids and multiply to form clone; any transformed bacteria are identified using genetic markers.
- Genetically engineered bacteria transcribe and translate the human gene to make human insulin.
- Human insulin is extracted and purified so it can be used in humans.
(o) Outline the process involved in the genetic engineering of ‘Golden RiceTM’;
Golden Rice was genetically engineered to control vitamin A deficiency in parts of the world where rice is a staple food. Vitamin A is present in the outer layers of rice, but in tropical conditions this layer is removed to stop harvested rice from rotting. Vitamin is not present in the remaining endosperm.
- The psy gene from a daffodil and the crtl gene from the soil bacterium Erwinia uredovora are isolated using restriction enzymes.
- A plasmid is removed from the Agrobacterium tumefaciens bacterium and cut open with the same restriction enzymes.
- The psy and crtl genes and a marker gene are inserted into the plasmid.
- The recombinant plasmid is put back into the bacterium.
- Rice plant cells are incubated with the transformed A.tumefaciens bacteria, which infect the rice plant cells.
- A.tumefaciens inserts the genes into the plant cells’ DNA, creating transformed rice plant cells.
- The rice plant cells are then grown on a selective medium; only transferred rice plants will grow because they contain the marker gene that’s needed to grow on this medium.
The resulting rice plants produced seeds with β-carotene in the endosperm, which is yellow, hence ‘golden’. Vitamin A is produced in our bodies from β-carotene.
