Module 16 - Model Organisms and Genetic Frontiers Flashcards
Describe the priciples and features of Sanger dideoxy-sequencing and what a dideoxynucleotide is.
Saber sequencing uses bases that terminate polymerisation because they lack a 3’-OH (dideoxynucleoside triphosphate).
Each DNA samples are replicated and labelled with a primer. It is then added with the four bases, DNA polymerase, as well as the dideoxynucleoside triphosphate of one of the base type. The reaction is repeated for each ddNTP of the other bases.
Each reaction produces a pool of DNA molecules that end in a particular base. If you combine all 4 pools, a complete set of all possible truncations of the original DNA molecule would be obtained as when running on a gel, the position of the truncations of the gel would depend on the length of the base section.
Describe the principles and features of Illumina Next-Generation Sequencing (NGS) and how it compares with Sanger sequencing.
NGS uses a similar principle as the Sanger sequencing as they use bases with a 3’ block (terminating sequence) as well as distinct fluorescence dye.
However, unlike Sanger method where each gene fragment needs to be sequenced individually, NGS allows concurrent sequencing of millions of fragments at the same time.
As the four modified nucleotides are added to the segment sequence, it is imaged before the dye and the terminating group is cleaved. This allows for other fluorescent bases to be added to the 3’ end where the process repeats.
Describe the principles and features of nanopore (minION) sequencing
Nanopore passes an ionic current through it and measures the changes in current as biological molecules pass through. The information about the change in current can be used to identify that molecule.
A strand of DNA is passed through a nanopore, where the current changed as the G, A, T, C bases pass through the pores, identifying them.
Explain what the purpose of the ENCODE project is and give examples of the types of genomic data it is generating.
Its purpose is to identify all functional elements in the human genome (not just the sequence), which may include generating data such as:
- expression levels of transcripts
- transcription factor binding sites
- histone mark enrichment
- open chromatin
- chromosomal 3D arrangements
Describe the concept of gene therapy and give an example in regards to haemophilia.
Gene therapy refers to the use of recombinant DNA to treat a disease or disorder by altering the genetic makeup of the patient’s cell (replacing the faulty copy of the gene).
For example, in treating Haemophilia, they have been testing the usage of Adenovirus-Associated Virus (AAV) to carry the Factor IX gene into the patient’s cell.
Explain the central reasons why model organisms are useful for biomedical research.
Ity involves a balance between relevancy and utility.
Utility refers to the ease of use (cheap and quick), powerful genetic tool of model organisms in manipulating and visualising gene expression.
Relevance of the model organism to humans due to evoltionary conservation of genes, molecular pathways, cellular behaviours, and developmental processes.
Mention the popular deveopmental model organisms.
Worm, vinegar fly, zabrafish, clawed frog, chick, mouse
Mention methods of expression analysis in Drosophila development.
It involves two main topics:
- Where is the gene transcribed?
- In-situ hybridisation staining (detect mRNA)
- gene reporter
- Where is the protein (gene product) expressed?
- immunohistocehmical staining
- fusion proteins
Mention an example of gene reporter.
Reporter gene is attached to a regulatory sequence of another gene of interest.
Certain genes are chosen as reporters because the characteristics they confer are easily identified and measured, or because they are selectable markers.
Mention a method to conduct functional analysis of Drosophila development.
It involves loss of function analysis: examining homozygous mutant cells/embryos
An example of this is the GAL4/UAS transcriptional activation binary system. GAL4 binds to UAS and activates expression of downstream genes.
If the systems are separated between individuals, then the GAL4 and UAS lines would only come together in the F1 progeny.
