3.1.6 Characterisation of genes and gene products at a molecular level (methods) Flashcards
What are the main three types of gene analysis methods?
Hybridisation based techniques
Enzyme based techniques
Use of antibodies
What are the fundamentals of hybridisation?
Nucleic acids hybridise (joining of two complementary strands, annealing) to complementary strands
Double stranded molecules can be denatured/separated by heat
Complementary sequences re-nature (re-hybridise) at lower temperatures
Name the denaturing agents
Heat, NaOH
How can particular sequences be detected using hybridisation?
Through the use of fluorescently labelled probes.
A sample of DNA is denatured using heat and NaOH
A surplus of GFP labelled probes (oligonucleotides, short sequences of nucleotides) is added to the sample, if complementary strand is found then the probe will anneal. These compete with the other complementary strand.
The sample is washed through and any excess probes are also removed
If the target gene is present, the probe will bind to it and be able to be detected.
How are strands allowed to re-anneal?
The solution is cooled, so hydrogen bonds are allowed to reform
What is fluorescence in situ hybridisation (FISH)?
This is where (long) probes of DNA are labelled with fluorescent dye and then are allowed anneal to DNA through denaturing the chromosomes and then allowing the probes to hybridise.
What is fluorescence in situ hybridisation (FISH) used for?
This is for identifying where regions of DNA are on different chromosomes, so is useful for indicating translocations - for example, the ‘Philadelphia chromosome’, where chromosomes 9 and 20 undergo a translocation mutation
How can ‘in situ’ approaches like chromosome painting be useful?
This allows the ‘painting’ of the entire karyotype (arrangement of chromosomes) - in this method, all of the chromosomes can be uniquely and entirely coloured/labelled, allowing chromosomal abnormalities (such as translocations) to be easily recognised
How can RNA interference (RNAi) mechanisms be exploited to regulate gene expression?
Exogenous (externally derived) siRNA molecules that are complementary to the target gene can be inserted into the cell using lipid capsules, where they will associate with a RISC factor and inhibit gene expression
What are miRNAs and siRNAs, and how do they function?
These are micro RNAs (miRNAs) and short interfering RNAs (siRNAs), short sections of nucleotides that are associated with RNA induced silencing complexes (RISC). The miRNAs are complementary to short sequences on mRNA, so bind to those molecules and therefore guide the RISC complex. This will then cause the mRNA molecule to be degraded or its translation to be inhibited.
siRNAs act in the same way but are derived from a different molecule.
- Clinical application of siRNA approaches
Still in trial stages (and progress has been slower than anticipated), but has been shown to lower transthyretin levels in patients.
Transthyretin (TTR) is formed in the liver, and deposits of this protein in the heart and kidneys can cause amyloidosis (build up of abnormal transthyretin amyloid proteins in cells causing abnormal shape, eventually can cause organ failure). Most common mutation in TTR causing misfolding of fibril proteins
RNAi methods have been used to target the production of transthyretin mRNA, therefore suppressing deposition and reducing production of these proteins.
What does CRISPR stand for?
Clustered regulatory interspaced short palindromic sequences
What enzyme is associated with the CRISPR complex?
CAS9 nuclease
Where does the CRISPR machinery originate from, and what has it been repurposed for?
It originates from microbial immune systems but has been repurposed to aid eukaryotic gene editing systems
What function does CAS9 have?
It is a nuclease, so once directed to a certain region of DNA by guide RNAs it is able to cut the DNA strands
What is CRISPR machinery able to do?
It can add or knock out specific genes at any region along the genome (if correct guide RNAs are used) although due to the protective nature of DNA it can be difficult to get exactly what is needed into or out of the genome
What is homology-directed repair?
This is a method for gene knock-in, where CRISPR machinery causes a break in the genome and then a DNA template located next to the double stranded break in the original DNA strand is able to ‘invade’ the neighbouring strand at this point of breakage and utilise the cell’s own DNA repair systems to form a complementary strand, for both the original and the template (see diagram in Andre Furger lecture).
In this way, the gene is inserted into the genome.
What is non-homologous end joining?
This is a method for gene knock-out, where after CRISPR machinery has removed a sequence from the strand, leaving non-matching sticky ends, and the cell’s natural repair machinery causes the DNA to be repaired in an error-prone manner, preventing the gene from being translated. New nucleotides are added or the non-matching bases are excised, sometimes resulting in a frame shift mutation, but the end result is that the gene is unable to be transcribed/is ‘switched off’.
List the 6 ways in which CRISPR-CAS9 complexes can be used to manipulate genomes:
- Induce a mutation
- Insert or replace sequences
- Large deletions or rearrangements (i.e. translocations or inversions) through use of pairs of gRNA directed CAP9 nucleases
gRNA-directed CAS9 can be fused to:
- Activation domains on DNA to mediate the upregulation/activation of specific endogenous (internal) genes
- Heterologous (different/not identical or complementary) effector domains to alter histone modifications or DNA methylation
- To DNA and also bind fluorescent proteins to enable imaging of specific loci along the DNA.
What are the three enzyme based techniques?
Restriction enzymes
Ligase enzymes
DNA polymerases
Restriction enzyme techniques are:
The use of specific nucleases, slightly different mechanisms to those seen in hybridisation techniques.
Seen in cloning and restriction fragment length polymorphism (RFLP)
Ligase techniques are:
Cloning and adapter ligation in sequencing
Ligase enzymes allow the formation of phosphodiester bonds between bases
DNA polymerase techniques are:
PCR (polymerase chain reaction) and sequencing
DNA polymerases are able to form strands of DNA
What are restriction enzymes (RE) able to recognise?
REs are able to recognise specific sequences of DNA at which they are able to bind and hydrolyse bonds between nucleotides.
These sequences are small (usually 6 nucleotides) and are often palindromic (complementary sequence reads the same in 5’ to 3’ direction of both strands)
There are indiscriminate ‘DNA scissors’ as well, but restriction enzymes are far more specific
What is electrophoresis?
This is where charged particles are separated out using a plate covered in an electrolyte and under the influence of an electric field.
How does DNA act in gel electrophoresis?
Due to DNA having a negative charge, it will move towards the positive electrode. Smaller fragments will be able to move faster through the electrolyte due to their smaller size/molecular weight, therefore allowing for fragments of DNA to be separated out by size.
How does restriction fragment length polymorphism (RFLP) work?
NOT COMMONLY USED ANYMORE
Restriction enzyme digests can be used to check for specific mutations, as if the mutation occurs at an RE recognition site or if insertions occur between two sites along the DNA then the results seen in electrophoresis (the restriction profile) will be changed due to different sizes of molecules.
