molecular biology Flashcards
what is the central dogma
- DNA makes RNA makes protein
- DNA makes RNA through transcription
- RNA makes protein through translation
what is molecular biology
the study of biological molecules, their reactions and interactions
what is a genome
total genetic content of a cell/organism
- the complete DNA sequence of a cell/organism
what is a transcriptome
all mRNA molecules currently present in a cell/organism
what is a proteome
total set of proteins currently present in a cell/organism
how are genomes studied
by transcriptomic and proteomic profiling
how do genomes vary
largely the same between cell types within an individual
how will transcriptomes vary
will vary between cells, and with environmental conditions
what can be used for the study of mRNA abundance
- RNA sequencing
- DNA micro assays
what can be used for the study of protein abundance
traditionally Ab based: ELISA assays
- needs antibodies and lacks highly parallel methods and universal external standard
- mass spectrometry
how will proteomes vary
will vary between types of cells and with environmental condition
what is important in proteomes
post translational modifications
- proteolytic cleavage
- covalent modification
what is mass spectrometry
method to accurately measure mass of proteins and protein fragments
what does mass spectrometry require
proteins/peptides are ionised to allow detection
what are the advantages of mass spectrometry
-high sensitivity, accuracy and resolution
what is the role of a mass spectrometer
generates gas phase ions from a sample, separates them according to their mass to charge ratio and generates a record of their abundance, allowing:
- MW determination
- amino acid sequencing
- PTM detection
- relative quantification
- structural analysis
what are some of the considerations of omic studies
- generate very large amounts of data
- can take a lot of time and money
- picking conditions in transcriptomics and proteomics may be critical
- risk of errors
- amount of data often exceeds amount of understanding
what is DNA sequencing used for
- nucleic acid sequencing can be used in diagnostics
- sequencing of genes amplified by PCR allows identification
give examples of next generation sequencing
- Illumina, SOLiD
Give an example of nanopore sequencing
minION
outline the drug discovery process
- target identification
- determine DNA and protein sequence
- elucidate structure and function of the target
- proof of concept and assay development
- assay for high throughput molecular screen
- high throughput screening/ directed synthesis programme
- select lead structures
how can drug targets be identified
- genetic association studies
- gene expression studies- transcriptomics
- protein expression studies- proteomics
what do genetic association studies involve
looking for presence of DNA sequence variation in diseased vs healthy individuals/cells
- identify mutations and polymorphisms associated with disease
what do gene expression studies involve
looking for differences in gene expression in diseased vs healthy individuals
what do protein expression studies involve
looking for differences in protein expression and modification in diseased vs healthy individuals
what properties does a good drug target need to have
- unique to pathogen/cell type
- otherwise higher risk of side effects - essential
- inhibition/antagonism needs to affect cell or tissue - non redundant (no other molecules with similar function)
- otherwise the other gene/protein may compensate - susceptible to modification
- drug like molecules need to modify activity
what are the 2 key steps involved in validation
- reproducibility- once identified via specific technique
- introduction of variation to ligand target environment
what should variation to ligand target environment lead to
- modulation of drug molecule activity results in changes in affinity for target
- variation in cell or tissue type should/should not vary effect
- mutation of binding domain of protein target should result in loss or modulation of drug effect
what is overexpression
making more of the protein than normal
what is gene knockout
deleting the gene and therefore stop protein production
how are animal models used in human disease
animal models can be genetically modified so that they develop human disease
- eg. knockouts, transgenes
what techniques are used in making the target
express recombinant protein in a suitable host
- E.coli
- Saccharomyces cerevisiae
- cells in culture (mammalian cells)
what are the advantages of using recombinant DNA techniques
- get large amounts of protein
- enables structural studies, functional studies and screening
- can make altered forms (change DNA to change resulting protein)
- study isolated domains/regions
- alter single amino acid residues and test hypotheses about drug binding
what is the genome project
- determined the DNA sequence for humans
- many G protein coupled receptor genes identified
- many of them of unknown function
what are downstream effectors
- if we know that a protein is overexpressed in a diseased tissue, whole cell transcriptome can elucidate the cascade that follows
- this can be used to identify potential drug targets
what are genetic polymorphisms
different in DNA sequence between individuals
- chance mutations
- induced mutations (viral infections, chemical agents)
what is a genotype
what is in the DNA
- inherited from parents
- deduced from gene sequence
give an example of a genotype
genes encoding eye colour
what is a phenotype
what is observed in an individual
- protein expression
- environmental factors
- epigenetics
give an example of a phenotype
an individual with brown eyes
what are the types of genetic polymorphisms
- single nucleotide polymorphisms
- copy number variants
- INDELS
what are single nucleotide polymorphisms
- most common type of polymorphism
- differ in base pair in DNA sequence
what are copy number variants
- segment of DNA can have multiple copies
- important in drug metabolism
what are INDELS
- insertion or deletion of a base pair
- often results in non functional protein
what are genome wide association studies
- collect very large data sets on individuals genetic makeup
- look for associations between genetic variations and different disease states
what is a Manhattan plot
- type of scatter plot
- helps identify which SNPs are linked to the condition
what are the clinical implications of genome wide association studies
- can establish correlation but not necessarily causation
- can be used for diagnosis and prognosis decisions
- starting point for further biological study
what are enteroviruses
responsible for several significant diseases (polio, common cold)
why is the common cold difficult to treat or prevent
rapid mutation impedes vaccine development
what is required for enterovirus pathogenesis
requires the host methyltransferase SETD3
- virus infects human cell
- uses host enzymes for replication
- cell bursts releasing new virus particles
describe the study for enterovirus pathogenesis
- studied the transcriptome of infected cells and worked out what proteins were being expressed
- produced a gene knockout strain of mice
- compared disease progression with wild type mice
what were the implications of this study
- not producing SETD3 enzyme appears to prevent virus replication
- gene knockout mice lived normally
what are pharmacogenetics
study of gene variations in drug response
