Chapter 21 Manipulating Genomes

What is a genome?

• All the genetic material an organism contains.

What is the main reason for screening and sequencing DNA?

• To identify and treat a range of medical problems.

• Knowledge of the genome cannot be obtained through the proteome because of the nature of human DNA (non-coding DNA), so other techniques have to be used to sequence it.

Explain the rapid advancements of Sangers sequencing techniques.

• Him and his team were originally able to sequence nucleic acids from viruses and bacteria through the radioactive labelling of bases and gel electrophoresis.

• It was originally carried out manually, including the human mitochondrial DNA.

• The techniques were developed further like swapping radioactive labels for florescent tags, which lead to scaling up and automation of the process.

• This lead to the capillary sequencing version of Sanger sequencing that was used in the Human Genome Project (HGH).

What method of DNA sequencing was developed as a result of Sanger sequencing?

• High-throughput sequencing has been developed based on Sanger’s original process.

• High-throughput sequencing is an automated, faster version that still uses coloured terminator bases to stop the reaction.

• This version is extremely efficient and is constantly being refined and developed.

What are terminator bases, and what are they used for in DNA sequencing?

• Terminator bases are modified versions of the four nucleotide bases (adenine, guanine, cytosine, thymine) which stop DNA synthesis when they are included.

What are the steps to DNA sequencing?

1. DNA is mixed with a primer, DNA polymerase, nucleotides and terminator bases.

2. Mixture placed in a thermal cycler that rapidly changes the temperature at intervals, at 96 degrees the DNA separates into single-strands, at 50 degrees the primers anneal to the DNA strand.

3. At 60 degrees DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single- strand.

4. When a terminator base is incorporated, it terminates the synthesis of DNA, no more bases can be added. This results in fragments of many lengths, depending on where the chain-terminating bases have been added during the process. After many cycles, all of the possible DNA chains will be produced with the reaction stopped at every base.

5. The DNA fragments are separated using gel electrophoresis according to their length.

6. Order of bases in the capillary tubes shows the sequence of the new complementary strand of DNA, which is used to build the original sequence of DNA. The data is fed into a computer which reassembles the genomes.

How has genome sequencing led to genome-wide comparison?

• Genome sequencing has made it possible scientists to compare the entire genome of individuals of the same species and different species. This has resulted in many advances including:

  • Identifying antibiotic-resistant bacteria.

  • Tracking the spread of pathogens to monitor potential epidemics and pandemics.

  • Identifying regions in the genome for new drugs to target.

• Comparing genomes has led to:

  • The accuracy of classification of species.

  • Our understanding of evolutionary relationships.

What is bioinformatics?

• The development of the software and computing tools needed to organise and analyse raw biological data.

• Includes the development of algorithms, mathematical models and statistical tests.

What is computational biology?

• Uses the data from bioinformatics to build theoretical models of biological systems, these can be used to make predictions about what would happen to the models in different situations.

• For example- it helps us to use the information from DNA sequencing to identify genes linked to specific diseases and to determine evolutionary relationships between organisms.

What is epidemiology and how are computational biology and bioinformatics contributing to this research?

• Epidemiology is the study of how often diseases occur in groups of people and why this happens.

• Computers can analyse and compare the genomes of many individuals and may reveal patterns in the DNA we inherit and which diseases we are vulnerable to.

What has gene sequencing taught us about evolutionary relationships?

• DNA sequences of different organisms can be compared and then they can calculate how long ago two species diverged from a common ancestor. Enables scientists to build up evolutionary trees.

How has genome sequencing led to the prediction of amino acid sequences?

• The relationship between phenotype and genotype is very complex.

• The DNA sequence of the genome should enable you to predict the sequence of amino acids it produces in all of the proteins it produces, however the evidence is that the sequence of the amino acids is not always what would be predicted from the genome sequence alone.

What is synthetic biology and what techniques does it involve?

• Broadly described as the design and construction of artificial biological pathways, organisms or devices or the redesign of natural systems.

• It includes:

  • Genetic engineering- a single change in the biological pathway or significant genetic modification of an entire organism.

  • Use of biological systems or parts of biological systems in industrial contexts.

  • The synthesis of new genes to replace faulty genes, like in treating cystic fibrosis.

  • The synthesis of an entire new organism.

What are introns?

• Non-coding DNA, they consist of many short tandem repeats (STRs).

What is the probability of two individuals having the same satellite pattern?

• Very low, only identical twins will have the same satellite pattern, the more closely related you are to someone the more likely you are to have similar patterns to them.

What is DNA profiling and what are its main uses?

• DNA profiling is the production of an image of the patterns in the non-coding DNA of an individual.

• Mostly used for assisting the identification of individuals or familial relationships.

What is the process of DNA profiling?

1. Extracting the DNA- this could be collected from a tissue sample, if it is a small fragment polymerase chain reaction (PCR) is used to give scientists enough DNA to develop a profile.

2. Digesting the sample- strand of DNA are cut into small fragments using enzymes called restriction endonucleases. These cut at specific points depending on the different types of the enzyme.

3. Separating the DNA fragments- the fragments need to be separated to form a clear pattern, for this electrophoresis is used. The DNA samples are loaded into wells and an electrical current is applied. DNA is negatively charged so the DNA samples move towards the positive end.

4. Hybridisation- florescent DNA probes are added in excess to the DNA fragments. They bind to the complementary strands of DNA.

5. Seeing the evidence- if radioactive labels were used, X-ray images are taken of the paper, if florescent labels were used it is placed under UV light. The fragments give a pattern of bars- the DNA profile.

Explain the process of PCR.

1. The DNA sample to be amplified, excess of nucleotide bases (in the form of deoxynucleoside triphosphates), primers, DNA polymerase are mixed in a vial and put into the PCR machine called a thermal cycler. The cycle can be repeated many times to produce many copies of the original DNA.

2. Separating the strands- temperature in the PCR machine is increased to 90-95 degrees for 30 seconds, this denatures the DNA by breaking the hydrogen bonds holding the strands together so they separate.

3. Annealing of the primers- temperature is decreased to 55-60 degrees and the primers bind to the ends of the DNA strands. They are needed for replication of the new strands to occur.

4. Synthesis of DNA- temperature is increased again to 72-75 for one minute, this is the optimum temperature for DNA polymerase. It adds bases to the primer, building up complementary strands of DNA and so producing double strands of DNA identical to the original sequence. The enzyme Taq polymerase is used which is obtained from extremophiles in hot springs.

What are the main uses of DNA profiling?

• PCR and DNA profiling is used on traces of blood left at crime scenes, the DNA profile is compared to a sample taken of a suspect or from a criminal database.

• Also used to prove the paternity of a child with in doubt.

• It can also be used to identify individuals who are at risk of developing particular diseases, certain introns have been associated with increased risk and incidence of some diseases.

What is genetic engineering?

• The manipulation of the genome of an organism.

What are the basic principles of genetic engineering?

• Isolating a gene for a desirable characteristic in one organism and placing it into another organism, using a suitable vector.

• An organism that carries a gene from another organism is termed ‘transgenic’ and is often called a genetically modified organism (GMO).

What are the basic steps of genetic engineering?

1. Isolating the desired gene- using the enzyme restriction endonuclease to cut the required gene from the DNA. The strands are cut unevenly with some being a few bases longer, this leaves unpaired bases called sticky ends. These sticky ends make it much easier to insert into a different organism.

2. Insert into a vector- most common vector is a bacterial plasmid but we can also use a bacteriophage or liposome. Once it enters the host cell, it can combine with the desired gene to form recombinant DNA.

3. The plasmid is cut using the same restriction endonuclease, it creates the same sticky ends which will be complementary to the gene fragment.

4. The enzyme DNA ligase is added to anneal them as it catalyses the condensation reaction to form the phosphodiester bonds.

5. The vector then needs to be inserted, to do this the cell surface membrane must be more permeable. This is done through electroporation which makes the membrane more porous.

How can we identify transformed cells in genetic engineering?

• We use marker genes to identify which bacteria successfully took up the recombinant plasmid. The different marker genes are:

  • Antibiotic resistance genes

  • Genes coding for fluorescent proteins

  • Genes coding for enzymes

How has soya been genetically modified?

• One modification is the addition of a gene that produces the Bt protein, which is toxic to pests.

What are the ethic concerns surrounding GM soya?

• Positives:

  • Reduced the need for farmers to use pesticides

  • Results in a much higher yield

  • Longer shelf life to reduce food waste

• Negatives:

  • The genes might spread to the wider environment

  • People may be allergic to the different crops

  • The technology is often patented and therefore very expensive for poorer farmers

What are some issues relating to patenting and technology transfer?

• Patenting an item means that no-one else can use it without payment.

• The people who need the benefits of drought-resistant crops for example may not be able to access them because they are too expensive.

What is pharming and what are its potential ethical issues?

• Pharming is the use of genetically modified animals for pharmaceuticals.

  • Creating animal models- addition or removal of genes so that animals develop certain diseases

  • Creating human proteins- introduction of a human gene coding for a medically required protein

• Ethical issues include:

  • Welfare concerns

  • Religious beliefs

  • Increased risk of transmission of disease between species

What is genetic engineering in microorganisms?

• Pharming is a big use of genetic engineering in microorganisms, this is when a human gene is inserted into a bacteria so that they produce a human protein.

• Ethical concerns:

  • Increasing antibiotic resistance in bacteria

  • Increased risk of cancer in patients receiving the vectors due to DNA insertion causing mutations

What is gene therapy?

• When human DNA is altered to treat disorders.

What is somatic cell gene therapy?

• Replacing the mutant allele with a healthy allele in the affected somatic (body) cells.

• Viral vectors are often used for somatic cell therapy, with the desired allele inserted into its DNA.

• It is only a temporary solution for the individual as the somatic cell has a limited life and are replaced with cells that have the faulty allele.

• Therefore the faulty allele will be passed on to the individuals children.

What is germ line cell gene therapy?

• Inserting the healthy allele into the early embryo as part of in vitro fertilisation (IVF).

• The individual would be born with the healthy allele into its place and would pass it onto their offspring.

• This is illegal in humans due to ethical and medial concerns.