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Linda's Flashcards

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Biology 101 flashcards
1
Q

Population Genetics

A

Study of changes in the genetic composition of a population that occur over time and under evolutionary pressures

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2
Q

Genetic markers

A

-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.

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3
Q

Genetics of Lactase Persistence/tolerance

A

-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

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4
Q

Lactose intolerance

A
  • 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)

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5
Q

SNP

A
  • Single nucleotide polymorphism

- A single nucleotide locus with two naturally existing alleles defined by a single base pair substitutions.

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6
Q

LP genetic markers

A

-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

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7
Q

Allele frequency

A

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)

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8
Q

HWE

A

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

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9
Q

Principles behind HWE

A

To reach equilibrium, 5 strict conditions must be met:

  1. Large population of randomly breeding individuals
  2. No natural selection
  3. No new mutation
  4. No migration
  5. No genetic drift
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10
Q

Mendelian disease

A

-Single gene, trait controlled by a single locus
-Mutation in one gene
-LP is a mendelian disease
CC, CT=LP
TT= lactase intolerant

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11
Q

Pesticide resistance

A
  • 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.
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12
Q

Pesticide example

A

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

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13
Q

Pain candidate gene

A

-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
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14
Q

Genome editing

A

Insertion or deletion of DNA through engineered nucleases

using enzymes

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15
Q

Zinc finger proteins (ZF)

A

-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

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16
Q

Benefits of ZF nucleases

A
  • 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.
17
Q

How ZF nucleases work

A
  1. Design a nucleotide sequence, then put it in the cell by transfection or electroporation
  2. 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.
  3. 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

  1. DNA binding domain=will recognise the target you are targeting in the genome sequence you are editing
  2. DNA cleaving domain=recognises restriction enzyme site of FokI
18
Q

ZF:

Non homologous end joining

Homologous recombination

A

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

19
Q

Meganucleases

A
  • Derived from microbial mobile genetic elements
  • Integrates nuclease and DNA binding domains
  • Not widely used now
20
Q

TALENS

Transcription activator like effector nuclease

A
  • Used to modify the genome of any organism
  • Can induce mutation (via NHEJ) or insert DNA
  1. Identify target sequence
  2. TALEN sequence is engineered
  3. TALEN in inserted into a plasmid
  4. DNA transcription to produce mRNA
  5. mRNA translated to produce the functional TALEN
  6. TALEN binds and cleaves target sequence
  7. Introduction of error or new DNA sequence
21
Q

TALEN example

A

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
22
Q

TALEN example

Chimeric Antigen Receptor T cells (CAR T-cells)

A
  • 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
23
Q

CRISPR

A

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
24
Q

Delivery of CRISPR

A
  1. Viral delivery e.g. adenovirus (dsDNA0, AAV (ssDNA), Lentvirus (RNA)
  2. Lipid nanoparticle delivery
    - chemical method
    - DNA is coated in lipid, making it easier for it to move across the membrane
  3. Direct nucleic injection e.g. plasmid
25
Q

Genome editing tools

A

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.

26
Q

CRISPR

A
  • 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.
27
Q

Manipulating CRISPR

A
  • 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.

Advance Genetics (8 decks)

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