Module 6 Manipulating Genomes

Question 1

DNA Sequencing

Answer 1

are referred to as the Sanger method or chain termination method. Developments have led to high throughput sequencing, which is much faster, using capillary electrophoresis. Next generation sequencing uses specialised programmes and is automated.
* Chain termination sequencing uses dideoxynucleotides, which pair with nucleotides on the template strand of DNA and stop DNA polymerase from carrying out DNA replication.
* 4 test tubes are set up, each containing DNA primers, DNA polymerase, free nucleotides, one type of dideoxynucleotide (A,T,C,G), the DNA to be sequenced in single strand form. The test tubes are incubated at a temperature suitable for DNA polymerase to function. DNA primers anneal to form double stranded DNA and then DNA polymerase carries out replication, forming hydrogen bonds between free nucleotides and the template strand. Random insertion of dideoxynucleotides leads to termination of strand at a known base. DNA strands form that vary in length. Developing strands are separated from the template strand. Gel electrophoresis is then used to separate the developing strands. Shorter strands will move furthest along the plate.
* High throughput sequencing uses dideoxynucleotides labelled with fluorescent tags. Strands are separated by mass using capillary electrophoresis. It is very high resolution and a laser beam and detector read the colour and position of tags. It is high speed so can be used to sequence the whole genome of many organisms. Used to show evolutionary relationships. Next generation sequencing such as nanopore sequencing is faster and can reduce costs.

Question 2

• Comparing Genomes

Answer 2

• A genome is all the genes present within an organism. This can be compared between individuals or species.
• An organism’s genome can be used to sequence the amino acid sequence and from that the tertiary structure of the protein.
• Bioinformatics is used to generate data of sequences and the relationship between genotype and phenotype. High powered computers store databases of organism’s genomes and can be compared to other genomes. This can be used to determine how closely related species are or to use genomes as model organisms for humans. If genetic variation is present, there will large numbers of differences in the base sequence.
• If organisms have similar genomes, they share a more common evolutionary ancestor. The protein cytochrome c is used as it is found in most organisms and used in respiration.
• Interactions between genotype and phenotype can be shown through knocking out genes and targeting specific base sequences, as the effect on phenotype can then be observed.
• Epidemiology – the study of the spread of infectious diseases. The genomes of pathogens can be sequenced used to develop antigens for vaccines and methods of control.
• Human genome project was a publicly funded research programme that sequenced the entire human genome.
• Non-coding DNA is present in the genome. Through alternative splicing and post-translational modifications, it is difficult to sequence the human proteome from the genome.
• Synthetic biology creates new pathways by altering an organism’s genome, so it operates in a novel way. Bacteria cells used to synthesise human insulin.

Question 3

DNA Profiling

Answer 3

• Genetic fingerprinting is used to identify suspects and bodies. It can be used to assess the risk of developing diseases and conserving species.
• Organisms has short repeating units of 20-50 bases called VNTRs (variable number tandem repeats), which are unique to everyone.
• DNA is obtained from body cells and amplified using PCR. Restriction endonucleases cut DNA molecules into fragments. These fragments are separated using gel electrophoresis. Fluorescent/ radioactive probes that are complimentary bind to specific VNTRs and X-Ray/ UV light is used to produce a pattern.

Question 4

PCR

Answer 4

• Polymerase chain reaction is a form of in vitro DNA amplification which produces a large quantity of identical DNA or RNA fragments.
• The requirements for PCR include the target DNA, primers that anneal to the 3’ end, Taq polymerase which won’t denature at high temperatures, free nucleotides and a buffer solution.
• Process: during each cycle the amount of DNA is doubled. Denaturation is when the DNA is heated to 95 degrees which breaks the hydrogen bonds between the DNA strands. For annealing the temperature is decreased to 55 degrees and the primers anneal to the 3’ end of the DNA strands. In elongation/extension the temperature is increased to 72 degrees and Taq polymerase builds up the complimentary strand.

Question 5

Genetic Engineering

Answer 5

• Genetic engineering is the manipulation of gene sequences in an organism. As the genetic code is universal, DNA from different species can be combined to form recombinant DNA in a transgenic/GM organism.
• Recombinant genes are formed when fragments of DNA are transferred from one species to another.
• Genetic engineering can be used to increase resistance to diseases, increase yield or modify bacteria cells to produce insulin.
• Techniques- the desired DNA fragment is identified and isolated. This fragment is multiplied using PCR and then transferred using a vector (plasmid, virus or lipase). Electroporation is used to encourage uptake of plasmid vectors.
• Enzymes – restriction endonucleases cut genes at specific base sequences. Ligase joins together sticky ends of DNA. Reverse transcriptase builds up a double strand of DNA. Markers are used to track organisms including fluorescent markers, antibiotic resistance and enzyme markers.
• Genetically modified organisms are used as it is much faster and reduces costs.
• Genetically modified soya inserted with the Bt-toxin gene produces insecticide which creates alkaline conditions that kill insects .
• Pharming – livestock have been modified to produce pharmaceutical drugs.
• GM pathogens can be used to aid development of drugs. Adenoviruses can be altered to act as vectors for gene therapy.
• Ethical issues – biotech companies patent GMOs and can charge farmers more money. Lack of long-term research into health risks from food. Could contaminate non-GMOs, reduce biodiversity and invade natural habitats.

Question 6

Gene therapy

Answer 6

• Gene therapy alters the genetic material of an individual to treat or cure a disease. This can be done by replacing, inactivating or inserting a new gene. Vectors are used in the delivery and can be viruses or non-viral such as liposomes.
• Somatic cell gene therapy targets specific body cells, and so won’t target gametes. The effects may be short lived. Ex vivo therapy involves blood or bone barrow cells being extracted from the body and exposed to virus vectors. The new gene is inserted into the cells and then cells are grown in the lab, before being returned to the body via an injection into the vein. In vivo therapy is when the new gene is inserted into the cells by a vector inside the body.
• Germ line cell gene therapy involves genetic material being inserted into early embryos or gametes. It is illegal in humans are the changes may be permanent and affect future generations.