Genetic strategies Flashcards
(23 cards)
Define ‘Forward genetics’
Approaches which seek to find the genes encoded by DNA that are responsible for a phenotype of interest
(Phenotype —-> Genotype)
Define ‘Reverse Genetics”
approaches which seek to find the phenotypes linked to specific sequences of DNA
How is reverse genetics carried out?
Through complete knockout of the gene function or altering its sequence
What are the 3 main steps associated with forward genetics
1) Isolate mutants with phenotype of interest
2) Clone/identify the genes
3) Analyse the genes to predict encoded genes
List a few examples of good genetic model organisms (latin names)
- Escherichia coli
- Saccharomyces cerevisiae
- Drosophila melanogaster
What are the 3 types of mutagens
- Physical
-Chemical
-Biological
Give examples of physical mutagens
UV
ionising radiation (X-rays, gamma rays)
Give exmaples of chemical mutagens
- base analogues eg. 5-BU
- alkylating agents eg. ENU,EMS,MMS
What is a base analogue?
A chemical compound which closely resembles the nitrogenous bases found in DNA
How does a base analogue cause mutagenesis, giving an example
During DNA replication, the base analogue is mistaken for DNA base, this disrupts base pairings leading to base substitution mutations. For example 5-BU , resembles Thymine, so mispairs with Guanine, causing a transition mutation from AT —> GC
Define mutagenesis
Mutagenesis is the process by which the genetic information of an organism is altered, resulting in a mutation
How do alkylating agents cause mutagenesis?
- Through adding alkyl groups to nitrogenous DNA bases, forming abnormal base pairs, leading to mutations during replication
- Can also cause single/double stranded breaks in DNA if the modifications destabilise the sugar phosphate backbone
-Intra and Inter strand crosslinks form which interfere with DNA replication and transcription
Give some examples of alkylating agents
ENU, EMS, MMS
How do biological mutagens cause mutagenesis?
Transposable elements (transposons) are DNA sequences that can move from one location in the genome to another, disrupting genes or regulatory regions, leading to deletions, duplications or inversions of genomic regions
Describe how screening was used to test for temperature sensitive mutants
- mutant protein is functional at permissive temperature, whereas non-functional at restrictive temperatures
- for example in yeast cells, mutagenized cells proliferate and form colonies at 23 degrees, but not 36 degrees , two identical plates are incubated at these temperatures and missing colonies gives information about temperature sensitive mutation
How was screening for temperature sensitive mutants used in S. pombe?
- S pombe strains at different temperatures are blocked at different phases of the cell cycle
- Cell length and septum formation are very good indicators of cell cycle stage
How can gene function be restored in mutants
- clone mutants
- transform cells with gene/cDNA library
- isolate transformants which restore a wild-type phenotype
What is a gene library?
a collection of recombinant clones
Describe the two types of gene libraries
Genomic Library - contains DNA fragments that represent the entire genome of an organism (containing exons and introns)
cDNA library - contains DNA that is complimentary to mRNA expressed in a specific tissue or cell type
Explain how a genomic library is constructed (5)
- extract DNA from organism
- fragment the DNA using restriction enzyme
- insert the DNA into vectors eg. plasmid
- introduce the recombinant vectors into host cells via electroporation, transduction or transformation
- grow the host cells to create a population each containing a different DNA fragment
Explain how a cDNA library is constructed
- RNA is extracted
- reverse transcriptase makes a DNA copy
- treated with RNAse H
- make the 2nd strand of DNA with DNA polymerase, clone cDNA into suitable vectors
- transform host cells to create a library
What tool can be used in protein prediction?
BLAST - Basic Local Alignment Search Tool
- predicted protein sequence is compared with database sequences , giving information about function that can be tested
