6.1.3 - Manipulating Genomes Flashcards
What is DNA sequencing?
Finding a nucleotide sequence for a gene or the whole genome
What are the 2 types of DNA sequencing?
- Sanger sequencing
- High-throughput sequencing
What are the 3 steps of Sanger sequencing?
- Create copies of DNA fragments by extracting a DNA sample and heating it to separate the 2 DNA strands. Then cut the DNA strands into DNA fragments and create many copies
- Create complementary DNA fragments by placing the DNA fragment in a mixture where DNA polymerases use DNA primers to attach to the DNA fragments and then make complementary DNA fragments by using DNA nucleotides. However if DNA polymerase uses a terminating DNA nucleotide instead it will stop it from adding further nucleotides. As a result many complementary DNA fragments are produced all ending with a different terminating DNA nucleotide.
- Analyse complementary DNA fragments by separating the DNA fragments by length and work out the original samples DNA sequence
What are the advantages of high-throughput sequencing?
- Automated
- Very rapid
- Cheaper
What are the 3 main benefits of DNA sequencing?
- It enables genome wide comparisons between individuals and species which reveals how closely related different individuals or species are
- Allows us to predict the amino acid sequences of genes to reveal the tertiary structure of the polypeptide the genes code for
- Useful for synthetic biology which modifies existing DNA sequences and these modified DNA sequences produce specific proteins which can then be used as drugs
What is bioinformatics?
Bioinformatics is a field of biology that involves the storage, retrieval, and analysis of data from biological studies. These studies may generate data on DNA, RNA and protein sequences, and on the relationship between genotype and phenotype. Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available which can help to find the degree of similarity between organisms which then gives an indication of how closely related the organisms are
What is gel electrophoresis?
A technique used to separate molecules of DNA, RNA or proteins
How are molecules of DNA and RNA separated?
By mass where shorter fragments have a lower mass and longer fragments have a greater mass
How are proteins separated?
- By mass determined by the size of their R groups or the number of amino acids present
- By charge determined by the R groups
How do you prepare a gel electrophoresis?
- Cut a line of holes called wells into a piece of agar gel
- Submerse the agar gel into a buffer solution
- Load the molecules you want to separate into one of the wells
- Place a negative electrode at the end of the gel with the wells and a positive electrode at the opposite end
- Apply an electric current moving from negative to positive electrode
- Multiple samples can be compared by filling multiple wells in the agar
- The different bands are seen by adding a fluorescent dye to the gel which glows under UV light
How do the molecules get separated in gel electrophoresis?
The lighter or more negatively charged a molecule is the faster it will move across the gel so in a given period of time these molecules will move further than those that are heavier or less negatively charged
What is gel electrophoresis used for?
- To separate DNA fragments for genome sequencing
- DNA profiling
What is genetic engineering?
The process of isolating a gene from one organism and placing it into another organism. These organisms can then translate the added gene because the genetic code is universal and we can use it to modify animals, plants and microorganisms.
What are the uses of genetic engineering?
- Gene therapy
- Modify plants such as soybeans to give them insect resistance
- Modify pathogens for research into developing new medical treatments
- Pharming where an animal’s DNA is altered so that they produce human proteins for medicine or they develop human diseases so new pharmaceuticals can be tested on them
What is gene therapy?
When a patient’s DNA is altered to treat or cure a disease
What are the 2 types of gene therapy?
- Somatic gene therapy
- Germ-line gene therapy
What happens in somatic gene therapy?
A new gene is introduced into body cells called somatic cells specifically targeting cells in the tissues that need the treatment. However as somatic cells eventually die this treatment has short lived effects.
What happens in germ-line gene therapy?
The germ is introduced into germ cells (sperm and egg cells). This means all the cells in the offspring will be altered and therefore the treatment has long term effects that will be inherited.
What are the ethical issues associated with genetic engineering?
- Should we modify animals to act as models for human diseases?
- Should we put human genes into animals?
- When a company genetically modifies an organism they will patent this modification meaning the company can charge a large amount of money for the modified organism. For example, they can charge more for genetically modified seeds which can prevent poorer farmers from successfully growing crops
What are the 2 ways DNA fragments can be produced?
- Restriction endonuclease
- Reverse transcriptase
How does a restriction endonuclease produce a DNA fragment?
It cuts the DNA molecule at a specific set of bases known as a recognition sequence
How does reverse transcriptase produce a DNA fragment?
It adds DNA nucleotides to an mRNA molecule which forms a new strand called complementary DNA. An enzyme then destroys the mRNA strand and a second DNA strand is built by DNA polymerase which results in a complete DNA fragment
What are the steps of genetic engineering?
- A DNA fragment containing the gene is prepared using restriction endonucleases that produce sticky ends
- The DNA fragment is inserted into a vector (often a plasmid) which is done by using the same restriction endonuclease on the plasmid producing sticky ends that are complementary to the DNA fragment. The DNA fragments nucleotides are then joined to the plasmid’s nucleotides by DNA ligase which forms a recombinant plasmid
- The recombinant plasmids are inserted into bacteria which is called transformation and requires the addition of calcium ions and heat shock or electroporation which makes it easier for the plasmids to pass through the bacteria cell surface membrane
- Identify the bacteria which do contain a recombinant plasmid by beginning genetic engineering with a plasmid that contains an antibiotic resistance marker gene so once placed in the antibiotic any bacteria with the recombinant plasmid die
- Multiply bacteria creating a lot of the DNA fragment
What are some of the issues during in vivo cloning?
- Sometimes the plasmid just rejoins to itself
- Sometimes the DNA fragments join to each other
- During transformation its likely that some bacteria just won’t take up a plasmid at all
