d1.3 (mutations & gene editing) Flashcards
in which direction does DNA polymerase synthesize?
5′ to 3′ direction
types of gene mutations x3
substitutions
insertions
deletions
what does gene GNAT2 control for? where is it found? how does it occur?
protein product named “transducing”, a protein required for colour vision
it is found on chromosome 1 complementary strand
single base pair substitutions
“Other times, substitution in DNA occurs and changes the amino acid that is translated from the RNA sequence generated.”
two examples & definitions of a substitution
Missense: single base pair mutation that changes the amino acid. If amino acid is similar to what it should me, functionally the protein can still work (perhaps not as well).
Nonsense: a single base pair mutation that changes codon to a stop codon. Results in incomplete primary sequence. Protein typically not functional (though depends on where in the sequence it occurs).
2 characteristics of glutamic acid (amino acid prior to sickle cell disease)
polar
mRNA = GAA
2 characteristics of valine (amino acid after to sickle cell disease)
non-polar
mRNA = GUA
if both copies of sickle cell gene are mutated, what occurs?
sickle cell disease
if only one copy of sickle cell gene is mutated, what occurs?
malaria resistance
how is malaria dangerous? how does sickle cell disease work against this?
attack red blood cells & causes fever symptoms
with sickle cell disease, your cells are too small and limits malaria’s asexual lifecycle
what causes huntington’s disease? on what chromosome is it located
CAG sequence normally repeats 10-35 times. in those with Huntington’s disease, it is repeated 36-120 times or more (gene HTT)
expanded sequence leads to the production of an way longer version of the HUNTINGTIN (yes spelt this way) protein, which then accumulates in brain cells and causes damage
occurs on chromosome 4
CCR5-Δ 32 involves deletion of what? what does it result in?
32 base pairs in the CCR5 gene
as this is not divisible by 3, results in frameshift
deletion results in a nonfunctional CCR5 receptor, which means the receptor is either not produced or is not expressed correctly on the cell surface
CCR5-Δ 32 immunity and vulnerability to a certain disease (1 each)
beneficial protection against HIV
makes people more susceptible to West Nile Virus
main causations of mutations x3
physical mutagens
chemical mutagens
DNA replication
example of chemical mutagen. what is it used for?
Benzene: carcinogenic. Used to make acetone, polystyrene, and nylon fibres.
how can ionizing radiation (x-rays and gamma rays) create mutations? x2
radiation with sufficient energy can knock one of more base pairs out of place.
ionizing radiation has enough energy to break a covalent bond, and enough to break an H bond.
examples of damages to DNA from radiation (name a few)
H bon breakage
double strand break
single strand break
base losses
base changes
dna cross linkage
protein cross linkage
pyrimadine dimer
look at slide 19 for a visual on proofreading
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in what region is mutation rates higher? why?
satellite DNA near the centromere, because it is the site where sister chromatids bind
mutations can also occur where? x2
mutations can also occur when? x1
in mitochondrial DNA, in RNA genomes
in transcription of RNA
define CpG site
any place where a cytosine is followed by a guanine
what is the methylation of cytosine?
sometimes causes cytosine to deaminated to thymine (look at slide 24 for visual)
define CpG islands
places with many CpG sites. higher chance of mutation
are we sure of what methylation does? what can it perform?
not sure what methylation does, in some places it regulates gene expression but hypermethylation often seen in cancers.
what is methylation essential for?
cell differentiation and embryonic development
mutations in somatic (body cells) are associated with what? does it pass on to the next generation?
cancer
not passed on to next generation
are mutations in germ cells passed on to next generation?
yes
how are new alleles formed?
mutation (when mutagens of errors in DNA replication change the base sequence of a gene)
examples of a neutral mutation x2
be the result of a silent mutation, in which the change to the DNA does not affect the structure or function of the encoded protein
affect traits that do not affect the survival or reproduction of the organism (such as having a free or attached ear lobe)
define knockout mouse. what are they used for?
are genetically engineered mice in which one or more specific genes have been deactivated or “knocked out.”
technique is crucial in biological and medical research, allowing scientists to study the effects of gene loss on development, physiology, and disease
define gene targeting
modifying the DNA of mouse embryonic stem cells to disrupt or delete a specific gene
3 steps to creating knock out mice
gene targeting: modifying the DNA of mouse embryonic stem cells to disrupt/delete a specific gene
embryonic stem cells: modified stem cells are then injected into mouse embryos (these embryos are implanted into a surrogate mother mouse)
breeding: offspring are screened to confirm that they carry the gene modification. these mice are then bred to produce a line of knockout mice where the targeted gene is consistently inactivated
types of knockout mice & short definition x3
Complete Knockout: The gene of interest is entirely removed or disrupted.
Conditional Knockout: The gene is knocked out only in specific tissues or at specific times.
Knock-in Mice: These are similar to knockout mice, but instead of disrupting a gene, researchers introduce a new gene or a mutated version of the gene into the genome.
applications of knockout mice x4
gene function (understand the role of specific genes in development, physiology, and behavior)
disease models (used to create models of human diseases)
drug development (evaluate the efficacy and safety of new drugs)
genetic pathways (understanding genetic pathways and interactions)
limitations of knockout mice x3
Compensation: Sometimes, other genes may compensate for the loss of the targeted gene, which can complicate interpretation of the results.
Complex Phenotypes: The phenotypic effects of knocking out a gene can be complex and may not always directly correlate with human diseases.
Ethical Considerations: As with any animal research, there are ethical considerations regarding the welfare of the animals used in research.
what does crispr stand for? what does it mean? what benefits do they have?
Clustered Regularly Interspaced Short Palindromic Repeats
sequences of DNA that are found in bacteria and act as a sort of immune system
they remember viruses that have attacked the bacteria before
what is crispr-cas9?
is a technique that allows scientists to make precise changes to the DNA in living organisms (think of it as a pair of molecular scissors that can cut DNA at specific locations)
what is cas9?
an enzyme that acts like a pair of scissors to cut DNA.
how does CRISPR-Cas9 work? x3 steps
Guide RNA: Scientists design a small piece of RNA called guide RNA (gRNA) that matches the DNA sequence they want to edit. This RNA guides the Cas9 enzyme to the correct spot on the DNA.
Cutting the DNA: The Cas9 enzyme, guided by the gRNA, makes a cut in the DNA at the targeted location.
DNA Repair: After the DNA is cut, the cell’s natural repair mechanisms kick in. Scientists can use this opportunity to add, delete, or change the genetic material at that location.
what does PAM stand for? what is its function?
Protospacer Adjacent Motif
it is a short, specific DNA sequence found next to the target DNA sequence that CRISPR-Cas9 wants to cut
what is the importance of PAM? x4
Recognition: PAM sequence is essential for the Cas9 enzyme to recognize and bind to the target DNA. (without the PAM sequence, Cas9 cannot locate or cut the DNA effectively)
Binding Site: The Cas9 enzyme, guided by the guide RNA (gRNA), searches the DNA for the PAM sequence. Once it finds this sequence, it binds to it and then the gRNA guides it to the exact location where the cut should be made.
Typical Sequence: In most commonly used CRISPR-Cas9 systems, the PAM sequence is usually a 2-6 base pair sequence immediately adjacent to the target DNA sequence. For the Cas9 protein from Streptococcus pyogenes (one of the most widely used versions), the PAM sequence is typically “NGG” (where “N” can be any nucleotide and “GG” are two guanines).
(watch a video on this cause wtf)
Variability: Different CRISPR systems and Cas proteins recognize different PAM sequences. This specificity allows for flexibility and customization in gene editing.
applications of CRISPR-Cas9 x3
Research: Helps scientists understand the function of specific genes by turning them on or off
Medicine: Potential to treat genetic disorders by correcting mutations in a patient’s DNA.
Agriculture: Can be used to create genetically modified plants with desirable traits, like disease resistance
advantages of CRISPR-Cas9 x2
Precision: Targets specific genes with high accuracy (can be tailored by PAM sequence, using different cas9 proteins).
Efficiency: Relatively easy and cost-effective compared to older methods of genetic modification.
ethical considerations of CRISPR-Cas9 x2
Germline Editing: Changes made to human embryos or germ cells (sperm/eggs) can be passed on to future generations, raising ethical concerns.
Unintended Effects: There might be unintended changes to the DNA, which is something researchers need to carefully monitor.
define conserved sequences. what do they indicate?
regions of DNA that show a high degree of similarity across different species.
this similarity suggests that these sequences have an important function that has been preserved throughout evolution
2 examples of conserved sequences
Gene Coding Regions: Exons (coding sequences of genes) can be conserved because the proteins they encode are critical for cellular functions.
Regulatory Elements: Sequences like promoters or enhancers that control gene expression can be conserved to ensure proper gene regulation.
define highly conserved sequences
a subset of conserved sequences that remain almost identical across a wide range of species. these sequences are extremely stable and show very little variation
2 examples of highly conserved sequences
Histone Genes: These genes encode proteins that are essential for DNA packaging in the nucleus. Their sequences are highly conserved across many species.
Ribosomal RNA (rRNA) Genes: These are critical for protein synthesis and are highly conserved across all domains of life.
