Functional genomics: study systems Flashcards
(42 cards)
Why are model organisms used in functional genomics?
- Phylogenetic relationships
- Homologous proteins with similar functions
- Apply conclusions to humans
Name some examples of model organisms
- Budding yeast
- Nematode worm
- Fruit fly
- Mouse
- Maize
Give some common features of model organisms
- Small - physical size and genome
- Known genetic and physical maps
- Rapid life cycle/reproduction and good offspring yield
- Easy to study under lab conditions
- Distinct mutant phenotypes
- Genetic resources etc available to use
What is the central dogma of molecular biology?
The linear and unidirectional transfer of genetic information from DNA to protein
What are the 5 key features of eukaryotic genes?
- Introns
- Exons
- Promoters
- Terminators
- 3’ and 5’ UTRs
Give some examples of additional features of eukaryotic genes
May not apply to all eukaryotic genes
- Additional promotors/terminators
- Remote control regions (enhancers/repressors)
- Operons
- Multiple reading frames
- Non-coding RNAs
What is the function of additional promotors/terminators?
Starts/ends transcription at different locations along the gene to give different length UTRs
What is an operon?
Several genes being under the control of a single promotor
Mostly applies to bacteria
What is a multiple reading frame?
DNA sequence containing overlapping genes read in different frames, or genes encoded in opposite directions
What is the role of a promotor and where is it found?
Upstream of a gene, required for transcription
What is the role of a terminator sequence?
Required to end transcription and involved in poly-adenylation
Give some ways in which mRNA can be modified to give different transcripts
- Alternative splicing
- mRNA editing (substitution/insertion/deletion/base modification)
- Trans-splicing
Insertion/deletion only seen in protozoa/viruses
What is alternative splicing?
Incorporation of different combinations of exons to produce different transcripts from the same gene
What is trans-splicing?
Joining exons from two different RNA transcripts. Results in chimeric RNA.
* Intragenic trans-splicing - mRNA from same genome locus but spliced from different strands
* Intergenic trans-splicing - exons from diverse genes
Which two processes are required for mRNA stability?
- 5’ capping
- 3’ poly-A tail
What is differential gene expression?
Variation of gene expression observed depending on source or response to a stimulus
E.g. different tissues, disease
Name 4 single gene approaches to analysing RNA expression
- Northern blot
- cDNA libraries
- PCR: real-time and reverse transcriptase
- RNA in situ hybridisation
Name 3 genome-wide approaches to analysing RNA expression
- Microarray
- RNA sequencing
- Long-molecule sequencing
What is an advantage of genome-wide approaches to RNA expression?
Relative to single-gene approaches
Large scale so can look for patterns of gene expression across many genes at once. Can be used to compare different conditions.
Describe the protocol for northern blotting
- Isolate total RNA/purified mRNA
- Separate by gel electrophoresis
- Transfer to nitrocellulose membrane - RNA becomes covalently attached
- Use ssDNA probe to look for sequence of interest
Describe the protocol for reverse-transcriptase PCR
- Produce cDNA from total mRNA using reverse transcriptase and a polyT primer
- Use gene-specific primers to amplify the cDNA
- Carry out standard PCR - product made if gene expressed
What are the advantages of reverse transcriptase PCR?
- Semi-quantitative (and can be coupled with qPRC)
- Sensitive to small samples
What is quantitative/real-time PCR?
Amplification is measured after each cycle. cDNA is fluorescently labelled to measure total amplicon produced. Based on the fact that quantity of initial template is proportional to how soon fluorescence is observed, and fluorescence is proportional to total amplicon produced.
What is the threshold cycle in RT-PCR?
Point at which fluorescence becomes observable and there is a rapid increase, indicating the start of the exponential phase. There is a linear/inverse relationship between Ct and initial template molecules.
