Manipulating genomes

What are the principles of DNA sequencing?

1. DNA for sequencing is mixed with primer, DNA polymerase, free nucleotides A, C, T, and G, and one of the four types of dideoxynucleotide (terminator bases)

2. The mixture is placed in a thermal cycler to separate DNA into single strands and allow the primer to anneal to the DNA strand.

3. Then DNA polymerase attaches to the primer and begins DNA replication using free nucleotides in the test tube.

4. At any time, DNA polymerase can insert a terminator base by chance, resulting in termination of DNA replication. This results in many DNA fragments of different lengths depending on where the terminator was inserted.

5. After incubation, the new complementary DNA strands are separated from the template DNA.

6. The resulting single-stranded DNA chains are separated by length by gel electrophoresis, and the base sequence can be built up one base at a time from this

7. The order of bases in the capillary tubes shows the sequence of the complementary strand of DNA.

What is high throughput sequencing?

• High throughput methods are automated and rapid, allowing for simultaneous sequencing of multiple DNA strands so produce large datasets very quickly

• Eg. flow cell sequencing

How has gene sequencing has allowed for genome-wide comparisons between individuals and between species?

• Bioinformatics and computational biology are contributing to research into :

  • Genotype-phenotype relationships - specific base sequences can be targeted to knock out different genes and observing the effect on phenotype

  • Epidemiology - genomes of pathogens can be sequenced to identify highly infectious strains + track progress of an outbreak

  • Evolutionary relationships - the genomes of different species/individuals in the same species can be compared. Species with a small number of differences will share a more recent common ancestor than species with a large number of differences

How can DNA sequencing and bioinformatics be used to increase the effectiveness a vaccination programme against ebola?

DNA sequencing

• High mutation rate means many strains exist

• Can predict strain

• So vaccine contains correct antigen

Bioinformatics

• Facilitates access to large amount of data

• Facilitates access to data on DNA and proteins

• Universal format

• Can identify source of outbreak

• Can identify vulnerable populations

• Vaccination program can target certain area

What is bioinformatics?

The development of software to analyse raw biological data

What is computational biology?

Uses data to build models of biological systems to make predications

How has gene sequencing has allowed for the sequences of amino acids in polypeptides to be predicted?

• The genetic code can be used to predict the amino acid sequence in a protein

• Once scientists know the amino acid sequence they can predict how the new protein will fold into its tertiary structure

How has gene sequencing allowed for the development of synthetic biology?

• Synthetic biology is the design and construction of new artificial biological pathways, organisms or devices, or the redesign systems that already exist in nature.

• A new genome can be assembled using existing DNA sequences or using entirely new sequences.

• eg. Genetic modification of organisms to produce specific drugs

What are the principles of DNA profiling?

1. DNA is extracted from tissue sample

2. PCR is used to produce large quantities of the fragment of DNA

3. Restriction endonucleases are used to cut the amplified DNA molecules into fragments at a specific nucleotide sequence called the recognition site

4. The fragments are separated using gel electrophoresis

5. An excess of radioactive or fluorescent probes are added which are complementary to specific VNTR regions and bind to them

6. X-ray images are produced or UV light is used to produce images of the fluorescent tags glowing. These fragments give a pattern of bars which is the DNA profile.

What are the uses of DNA profiling?

• Used by forensic scientists to identify suspects of crimes/to identify bodies

• Paternity tests

• Identifying individuals at risk of developing particular diseases - certain microsatellites have been found to be associated with increased risk of particular diseases.

What are the principles of PCR?

Polymerase chain reaction - in vitro method of DNA amplification which takes place in a thermal cycler.

1. Denaturation - double stranded DNA is heated to 95°C which breaks hydrogen bonds between the two strands

2. Annealing - temperature is decreased to 50-60°C to allow the primers to anneal to the ends of the single strands of DNA

3. Extension - temperature is increased to 72°C for at least a minute (optimum temperature for Taq polymerase) to allow DNA polymerase to add bases to the primer, building complementary strands of DNA and so producing new identical double stranded DNA molecules

Why is Taq polymerase used in PCR?

• It is obtained from thermophilic bacteria, meaning it doesn’t denature at the high temperature used in the first stage of PCR.

• The optimum temperature is high enough to prevent annealing of DNA strands that haven’t been copied yet

What is the method of electrophoresis to separate nucleic acid fragments?

1. Create agarose gel plate with wells

2. Submerge gel in electrolyte solution

3. Load the fragments into wells using micropipette

4. Apple electrical current to the tank. Negative electrode is connected to the end of the plate with wells to allow DNA fragments to move towards the positive end due to attraction between negatively charged phosphates of DNA and anode (positive electrode).

5. The shorter DNA fragments will move further from the wells than larger fragments

6. Fragments are not visible so transferred onto nitrocellulose membrane which is then heated to separate the two DNA strands. Probes are then added, after which an X-ray image is taken or UV light is shone to produce a pattern of bands which is compared to a control fragment of DNA.

How is gel electrophoresis used to separate proteins?

• The different amino acids determine the charge of proteins (due to different R groups)

• Buffer solutions are used to keep the pH constant as charge of R groups depends on pH

• Proteins are denatured to break disulphide bonds

• Used to show genotypes of individuals by separating polypeptide chains produced by different alleles

What are the principles of genetic engineering?

• Genetic engineering is the manipulation of the DNA sequences of an organism

• Isolation - of DNA containing required gene

• Insertion - of DNA into a vector

• Transformation - transfer of DNA into suitable host

• Identification - finding host organisms containing vector and DNA (containing recombinant DNA)

• Growth - of successful host cells

How is the desired gene isolated in genetic engineering?

• Isolation of desired gene

  • Uses restriction endonuclease to cut the required gene from DNA of an organism. Some restriction endonuclease make a clean cut in the DNA, however many cut the two DNA strands unevenly leaving sticky ends with unpaired bases which make it easier to insert the desired gene into the DNA of a different organism.

  • Another technique is isolating the mRNA for desired gene then using reverse transcriptase to produce a single strand of complementary DNA. DNA polymerase is then used to form double stranded DNA. This ensures there are no introns

How is recombinant DNA formed in genetic engineering?

• Vectors - used to deliver DNA fragments into a cell

  • Plasmids - once a plasmid is inserted into a new host cell it can combine with host DNA to form recombinant DNA. They often contain a marker gene such as antibiotic resistance which allows scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in growth media with antibiotic. Another marker used is GFP which fluoresces under UV light - if DNA fragment is inserted in between the marker gene then it will not function.

• To insert a DNA fragment into a plasmid, the plasmid is cut open using the same restriction endonuclease, resulting in the plasmid having complementary sticky ends to the DNA fragment.

• DNA ligase is used to form phosphodiester bonds between sugar and phosphate groups on the 2 strands of DNA, joining them together

How is the vector transferred into the host organism?

• Known as transformation

• One method is culturing bacterial cells and plasmids in a calcium rich solution and increasing temperature, causing bacterial membrane to become permeable so plasmids enter.

• Another method is electroporation - a small electrical current is applied to the bacteria which makes the membranes porous so plasmids move into the cells

What are positive and negative ethical issues relating to genetic manipulation of animals?

• Animals can be genetically modified to have desired characteristics such as increased muscle growth - this ensure all organisms contain the desired characteristic.

• Pharming

  • Scientists have also genetically modified livestock to produce pharmaceutical drugs in a process known as pharming. For example, sheep have been genetically modified to produce useful human proteins in their milk.

  • Animals have been used as models for development of new therapies by removing or adding genes so they develop certain diseases

  • Concerns about animal welfare during production of GM animals

What are ethical issues relating to genetic manipulation of plants and microorganisms?

• Soya plants have been genetically modified to produce their own insecticide, Bt protein. However this has led to insect populations becoming resistant to this toxin, reducing its effectiveness to protect crops.

• There are issues relating to patenting and technology transfer as biotech companies charge farmers more to use their genetically modified seeds. However, buying seeds year upon year can be a major struggle for farmers in developing countries meaning they may not be able to afford the GM seed. Patenting also prevents the harvesting of the seed from one year to the next.

• Microorganisms can be used to generated recombinant proteins and are used for research purposes and treatments. GM pathogens are also used for medical and epidemiological research - this process is less widely used and strictly regulated due to the concern of using pathogen for biological warfare.

What are the principles of gene therapy in medicine?

• Somatic cell gene therapy

  • Involves replacing the mutant allele with a healthy one in the affected somatic cells. This doesn’t prevent the disease occurring in the next generation and has to be repeated as the effects don’t last long due to somatic cells having a limited lifespan.

• Germ line cell gene therapy

  • The healthy allele is inserted into the germ cells - usually eggs or embryo immediately after fertilisation. The individual would be born healthy and would pass the allele to offspring. Not been done in humans due to ethical concerns.