D1.3: Mutations And Gene Editing Flashcards
What is a mutation?
Any change in the nucleotide sequence of base pairs in a DAN molecule
- can result in a new allele
- occur randomly and anywhere
Most mutations do not alter the polypeptide -> only alter slightly
- structure and function not changed
What is a gene mutation?
A mutation that occurs in the coding region (exon) of DNA (in a gene)
DNA base sequence determines the AAs in polypeptide -> gene mutations can lead to change in the polypeptide produced
Most mutations: harmful/neutral
Some: beneficial
Mutation in normal body cell -> not inherited
Mutation in gamete -> can cause genetic disease
What is a point mutation/base substitution?
When a mutation exchange one base randomly for a different one -> result in SNPs
- result in a change in the triplet -> change in codon
- many outcomes
-> because genetic code is degenerate -> change may not have an effect
What are SNPs? 300
Single nucleotide polymorphisms (SNP)
- represent a difference in a single DNA nucleotide
SNPs are normal in DNA -> occur once every 300 nucleotides (average)
- common in non-coding regions of DNA between genes
Can act as biological markers -> help locate genes associated with disease
What are the possible results of a substitution mutation ?
- Silent/same-sense mutation: results in a codon for the same amino acid
- no change in polypeptide -> degenerate code - Missense mutation: results in a codon for a different amino acid
- alters 1 AA in polypeptide
- ex: sickle cell anemia - Nonsense mutation: results in a stop codon
- polypeptide chain is incomplete -> affect structure and function
- ex: cystic fibrosis
What is an insertion mutation?
A nucleotide with a new base is randomly inserted into the DNA sequence
Changes AA that would’ve been coded for by original triplet -> new triplet
Can cause frameshift, nonsense or stop mutation
What is a frameshift mutation?
Involves their the addition of removal of a single DNA base, changing the reading frame
- affects every triplet/codon after the mutation
Dramatically change AA sequence therefore polypeptide
- function and structure
What is a deletion mutation?
A nucleotide is randomly deleted from the DNA sequence -> change in triplet/codon -> change in the AA it would’ve coded for
Changes every codon/triplet/AA after the mutation -> change in AA sequence -> change in polypeptide
- affect function and structure
Can result in frameshift, nonsense or stop mutation
What are the consequences of insertion and deletion mutation?
Usually considered more harmful than substitution -> usually frameshift mutation -> impact whole sequences after mutation
mRNA read in codons (group of 3) -> 1/2 nucleotides added or removed -> whole sequences shifted
Ribosome reads codons along mRNA but all shifted -> completely different AA -> completely different protein
- more/wrong AA, stop protein from growing
= whole DNA sequence read incorrectly
What are some external factors that can cause gene mutation?
Physical:
Radiation -> chemical changes in DNA
- high energy radiation (UV)
- ionizing radiation (X-ray, gamma ray, alpha particles)
Chemical:
Carcinogens: substances capable of causing cancer in cells
(All carcinogens are mutagens)
- bensoapyrene/nitrosamines in tobacco smoke
- mustard gas (chemical weapon, WW1)
Biological:
Infections agents
Human papilloma virus (HPV)
What are mutagens?
Mutagenic agents/mutagens: environmental factors that increase the mutation rate of cells
What are some internal factors that can cause gene mutation?
Can be produced at random during DNA replication/repairs -> errors in nucleotide sequence not detected by proofreading (by DNA polymerase)
-> usually when detected it is removed and replaced
Where in the genome are mutations more likely to occur?
Mutation hotspot: regions where mutations are more frequent
- ex: where cytosine is followed by guanine (CpG site)
-> methylation occurs -> C can become thymine -> substitution mutation - can occur repeatedly -> CpG island
- associated with certain cancers
Uncoiled DNA: more exposed compared to tightly coiled DNA
Non-coding regions of DNA
What are the consequences of mutation in germ cells?
Cells involved in inheritance of genetic information (egg, sperm, zygote) -> germ line
- use meiosis to produce gametes
- Mutation in germ cell -> can be passed on to offspring
Mutation in sperm -> affect zygote of offspring -> all cells developed from that zygote will have mutation
Female that inherit mutation -> contain mutation in germ cell -> passed onto future offspring
What are the consequences of mutation in somatic cells?
Somatic cells use mitosis to produce cells all over body -> grow into tissues/organs
Mutations common -> usually don’t cause cancer
- usually result in early cell death or destruction by immune system -> cell replaced
Somatic cell mutation -> associated with cancers
- not inheritable by offspring
- cancer result of uncontrolled mitosis (show importance of control) => tumor
Cancer starts when mutation occur in gene that controls cell division
- if mutated gene cause cancer -> oncogene
Explain how a mutation can lead to a new allele
Variation: result of small difference in DNA sequence between individuals
- result of mutation, meiosis, random fertilization
MUTATION: -> orignal source of genetic variation
Mutation in dividing cells of sex organs -> change in the gene of gamete -> passed to next generation
- essential for evolution by natural selection
- only source of variation in asexual species
New version of the gene -> allele:
advantage: more likely to be passed on (increase survival)
disadvantage: more likely to die out (decrease survival)
no effect
What was the human genome project?
International, collaborative research effort to determine the DNA sequence of the entire human genome
Completed in 2003
Important breakthrough -> scientist can research what each gene codes for and effect of gene on organism
- help prevention and treatment of diseases
What is gene knockout?
Genetic engineering technique
One way to discover function of a gene -> remove it from genome/make it unusable = gene knockout
Organisms with genes ‘knocked out’ -> knockout organism
- common ones are lab mice
- genetic library of knockout organism exist
Conditions that have been studied using gene knockout:
- obesity, diabetes, cancer likelihood, addiction, cardiovascular disease
What is gene editing?
Allow genetic engineers to alter the DNA of organisms by inserting, deleting or replacing DNA at specific site in the genome known to cause diseases
- allow accurate manipulation of the genome
NOT THE SAME AS TRADITIONAL GENETIC ENGINEERING
Which involves inserting a gene from one species to another
In past scientist used engineered viruses to insert genes
Involved in gene therapies (treatment of genetic disease by altering persons genotype)
- ex: treatment of cystic fibrosis or sickle cell anemia
What is CRISPR?
Most commonly used new gene editing technique:
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
Use natural defense mechanism of bacteria to cut DNA strands at specific points determined by guide RNA attached to enzyme (Cas9)
One cut -> scientist insert, delete or replace faulty DNA with normal DNA
What is a conserved/highly conserved sequence?
Conserved:
A section of DNA or RNA that shows minimal mutations overtime
- tend to be identical or similar across a species/group of species
Highly conserved:
Little to no mutations over long periods of evolution
Ex: those involved in DNA replication, transcription, translation, protein involved in cellular respiration
What are the existing hypotheses to explain conserved/highly conserved sequences?
Functional requirements:
Exist within genes that code for proteins that are essential for an organisms survival
- processes do not occur -> cell won’t survive
- minimize mutations -> not passed on
-> natural selection maintains by necessity
Slower mutation rates:
Certain sections of gene sequences are less prone to mutations -> mutation rate is much slower
- DNA repair/proofreading very active in coding region with high functionality
-> hypothesis claim that mutations in this area are more frequently spotted and corrected
Chernobyl nuclear accident
1986: nuclear reactor accident
Massive release of high energy radioactive isotopes in the environment -> 400x Hiroshima
All plant workers killed by fatal dose of radiation
Cloud of radioactive isotope spread all over Europe
Effect of nuclear fallout:
- Bioaccumulation -> high level of radioactive isotopes in fish (Germany, Scandinavia)
- More that 6k cases of thyroid cancer
- exclusion zone: 19 miles, will be safe for human habitation in 20k years
Miles of forest + animals + plants killed
BUT Lynx, owls, wild boars and other species -> began to thrive in exclusion zone
Example of knockout organisms: leptin in lab mice
Obesity research:
Knockout mouse (Lep<ob>) with mutated gene for hormone leptin -> used to deduce the function of this hormone</ob>
Leptin -> suppresses appetite when you are full
Without leptin mouse ate constantly
Other examples:
Cancer research:
Knockout mouse (P53) had disabled trp53 tumor gene
Cystic fibrosis:
- common data genetic disease in the USA
Knockout mouse (Cftr) had defect in the gene that makes CFTR, a protein that regulates the passage of salts and water in/out of cells
What is a short palindromic repeat?
Seuqnces that are used to target specific DNA sequences then delete/insert/replace a gene
What are the different molecules/part of DNA that are involved in CRISPR?
Guide RNA (gRNA):
A specially designed RNA sequence that directs the Cas9 protein to the specific DNA sequence to be edified
PAM (protospacer adjacent motif) sequence:
A short DNA sequence near the target site that helps Cas9 recognize where to cut (most common: NGG)
Cas9 protein:
Nuclease enzyme that acts like molecular scissors -> cut DNA at target location
Target gene:
Specific sequence of DNA that is being edited
What are the steps of CRISPR?
gRNA made that matches DNA sequences that will be modified
gRNA added to cell along with Cas9 enzyme
gRNA binds to the target gene and Cas9 cuts this DNA sequence
- PAM strand help Cas9 locate where exactly to cut
Insertion/deletion of nucleotides -> disrupts gene of interest
- results in a disrupted DNA
-> knockout organisms
-> Addition of gene from another organism
OR
Replacement of nucleotides -> corrects gene of interest
- results in a repaired DNA
-> allow mutated genes to be inserted -> effects suited
-> potentially used to treat genetic disease -> replace faulty with functional
