Linda's Flashcards
Population Genetics
Study of changes in the genetic composition of a population that occur over time and under evolutionary pressures
Genetic markers
-Identifiable segments of DNA sequence with a known physical location on a chromosome
-Maybe part of a gene or may have no function known e.g. SNP, VTNR, microsatellite
-Properties: easily identifiable, associated with a specific locus, highly polymorphic
-Purpose: to track inheritance of a nearby gene that has not yet been identified but whose approx location is known -> used in genetic analysis
To differentiate between individuals in a population
-Usually have a set of markers that amplify around the region
-Can either sequence that region to look for a SNP or if it has repeat regions, can analyse them using gel electrophoresis.
Genetics of Lactase Persistence/tolerance
-Autosomal dominant trait
-Enables life long digestion of milk sugar lactose
-Enzyme-lactase phlorizon hydrolase (LPH)
-Lactase persist into adult life in some but not in all
-LPH hydrolases the milk disaccharide lactose into its component monosaccharides, galactose and glucose for absorption in the small intestine.
-C –> transition
Located approx 14000 bp upstream of the lactose-phlorizin hydrolase (LCT) gene in intron 9 on chromosome 2
-SNP (T-13910) prevents down regulation of lactose activity after wearing. Affects a binding site of transcription factor
–> Lactose tolerance
Lactose intolerance
- If lactase is absent, the lactose cannot be absorbed by the intestinal mucosa
- Reaches the colon undigested where it is fermented by colonic bacteria
Symptoms: abdominal pain, diarrhoea, blotting
Due to recessive genotype (TT)
SNP
- Single nucleotide polymorphism
- A single nucleotide locus with two naturally existing alleles defined by a single base pair substitutions.
LP genetic markers
-Use genetic markers to PCR amplify a 111 bp target containing four LP SNPs
C/G, C/T, T/C, T/G
-Region sequenced to look for the SNP
-C/T-13910 means you have the dominant trait, lactase persistent
Allele frequency
Proportion of gene copies in a population that are of a given allele type
In a study where 18 individuals were genotyped, 72% were LP (CC), 44% CT and 28% TT.
Since its a dominant trait, high proportion of the population have the dominant allele (C)
HWE
Defines conditions in which the allelic and genotypic frequencies in a population are not changing over time
p2 + 2pq + q2= 1
The equation is used to calculate genotype frequencies based on allele frequencies
Principles behind HWE
To reach equilibrium, 5 strict conditions must be met:
- Large population of randomly breeding individuals
- No natural selection
- No new mutation
- No migration
- No genetic drift
Mendelian disease
-Single gene, trait controlled by a single locus
-Mutation in one gene
-LP is a mendelian disease
CC, CT=LP
TT= lactase intolerant
Pesticide resistance
- Disease persists in all living organisms due to changes in allele frequencies towards an evolutionary equilibrium in which mutations balance selection.
- The use of pesticides and antibiotics cause pests that were under control to return.
Pesticide example
DDT is an organochloride nerve toxin in insects. Common pesticide.
- Dominant mutations in a single gene confer resistance through detoxification of DDT
- In Bangkok, DDT uses resulted in increase in mosquito genotypes RR but these rapidly declined when stopped spraying
- Genotype RS decreased but then rapidly increased when spraying stopped. More advantage to have both alleles
- With insecticide application, strong selection favours heterozygotes.
R-dominant, resistance allele
S-susceptibility allele.
RR genotype confers a fitness cost. In the absence of the insecticide, resistance is subject to negative control
Pain candidate gene
-Genotyped and extracted DNA using a variety of genetic markers D18553, VNTR and SNP
VNTR:
- 20-100 bp sequence that is repeated up to thousands of times
- used to calculate the number of GC repeats
SNP
- genetic polymorphism within a population in which two alleles of the gene differ by a single nucleotide
- more common in genome and evenly distributed
- easy to locate
- deletion/insertion in the candidate gene creates short (s) alleles and long (L) allele
- Candidate pain gene has been cloned and mapped in human chromosome 17q
- Polymorphism has been identified and consists of different lengths of repetitive GC rich repeats sequences in the upstream regulatory region of the candidate gene.
- More repeats, higher pain tolerance
Genome editing
Insertion or deletion of DNA through engineered nucleases
using enzymes
Zinc finger proteins (ZF)
-small protein domains in which zinc plays a structural role contributing to the stability of the domain
-protein that recognises specific DNA sequence
-Structurally diverse functions
DNA recognition
RNA packaging
Transcriptional activation
Regulation of apoptosis
Protein folding
Assembly and lipid binding
Benefits of ZF nucleases
- Rapid disruption of or integration into any genomic loci
- Mutations made are permanent and heritable
- Works in a variety of mammalian somatic cell types
- Knock out/in cell lines in as little as two months.
How ZF nucleases work
- Design a nucleotide sequence, then put it in the cell by transfection or electroporation
- ZF will recognise the target sequence.
Have control over what genomic sequence you want to target and since you designed it, it will recognise the host genome and bind to it. - Once ZF binds, then have ability for restriction enzyme to cut. Have a protein not DNA bound dsDNA. Cuts DNA
DNA can join by NHEJ or homologous recombination
Two important domains
- DNA binding domain=will recognise the target you are targeting in the genome sequence you are editing
- DNA cleaving domain=recognises restriction enzyme site of FokI
ZF:
Non homologous end joining
Homologous recombination
NHEJ=DNA is broken, the DNA is removed and then the DNA will just rejoin using DNA ligase
Homologous recombination= can insert another gene/exon using homologous recombination
Meganucleases
- Derived from microbial mobile genetic elements
- Integrates nuclease and DNA binding domains
- Not widely used now
TALENS
Transcription activator like effector nuclease
- Used to modify the genome of any organism
- Can induce mutation (via NHEJ) or insert DNA
- Identify target sequence
- TALEN sequence is engineered
- TALEN in inserted into a plasmid
- DNA transcription to produce mRNA
- mRNA translated to produce the functional TALEN
- TALEN binds and cleaves target sequence
- Introduction of error or new DNA sequence
TALEN example
1yr girl with acute lymphoblastic leukemia
- bone marrow makes to many immature B cells
- > CD19 protein
- bone marrow transplant
- engineered immune cells that can seek and destroy cancer cells without harming the patient
- cancer of the blood, too much protein due to a genetic mutation
TALEN example
Chimeric Antigen Receptor T cells (CAR T-cells)
- Carries an antibody that tracks and kills any cells that make CD19
- CD19 is found on the surface of B cells, type of WBC
- TALENs used to cut a gene in the T cell that produces a protein called T cell receptor alpha chain
- That protein allows T cells to distinguish between a persons own cells and invaders
- Cutting out the gene means the T cells can no longer recognise anything as foreign.
- Stops patients body from rejecting the engineered CAR T-cells
- Then gave patient an antibody drug that kills that patients own T cells, letting the new donor cells grow
- 1 month after treatment, no signs of leukemia
CRISPR
Cluster Regularly Interspaced Short Palindromic Repeats
- Molecular scissors, cutting and replacing DNA letters in an organism’s genome with precision and ease
- RNA guided
- Precision DNA cutting
- Can edit multiple cells
Delivery of CRISPR
- Viral delivery e.g. adenovirus (dsDNA0, AAV (ssDNA), Lentvirus (RNA)
- Lipid nanoparticle delivery
- chemical method
- DNA is coated in lipid, making it easier for it to move across the membrane - Direct nucleic injection e.g. plasmid
Genome editing tools
Can use genome editing tools (ZFN, TAKEN, CRISPR-Cas9) to:
-Gene correction
-transcriptional regulation
-multiplex gene targetting
-gene knock out or report gene insertions
Achieve by homologous recombination and non homologous end joining.
CRISPR
- Based on the natural system used by bacteria to protect themselves from viral infections
- When bacterium detects the presence of virus DNA is produces 2 short RNA, 1 of which contains a sequence that matches that of the invading virus.
- These 2 proteins form a complex with a protein called cas9
- Cas9 is a nuclease, an enzyme that can cut DNA
- When the matching sequence (guide RNA) bonds to its target in the viral genome, the cas9 cuts the target DNA disassembling the virus.
Manipulating CRISPR
- Can be engineered to cut not just viral DNA but any DNA sequence at a precisely chosen location by changing the guide RNA to match the target
- If the guide RNA matches, cas9 cuts
- When this happens the cell will try to repair itself which can lead to mutations and the gene being deactivated allowing researchers to understand its function. Mutations are random
- But can be more precise. Can replace mutant gene with a new copy. Can add another piece of DNA that carries a desired sequence
- Once CRISPR system makes a cutm the DNA template can pair up at the cut ends, recombining and replacing the sequence with a new version
- Can be done in stem cells which can give rise to many different cell types and fertilised eggs
- CRISPR can be used to target many genes at once.
