technical methods- definitions Flashcards
basic principle of technical methods
choose the simplest, most direct method
PCR- what does it do
amplifies a specific DNA sequence determine day the experimenter. Specific and very sensitive
PCR uses
- Detection of viral and bacterial DNA in tissue samples
- Determining the size of a sequence of DNA between two ‘primers’
- production of DNA for making genetic tools (e.g. expression vectors) or DNA sequencing
qPCR- what does it do
quantifies a specific DNA (determined by the experimenter) sequence in a sample
qPCR uses
- quantifying viral load
- comparing amounts of DNA
- comparing amounts of specific mRNA (following conversion to DNA by RT)
RNA seq- what does it do
quantifies all RNA species in a sample- converts all RNA to DNA and then uses parallel sequencing to identify each molecule
RNA seq uses
comparative gene expression. Finding out which genes are regulated in a particular disease or in response to a specific stimulus. An un-bias method
DNA sequencing- what does it do
determines the base sequence of a specific stretch of DNA (sound 750 bases)
DNA sequencing uses
mutation detections, pathogen identification, gene identification
genome sequencing- what does it do
massive parallel DNA sequencing to establish the base sequence of the entire genome
genome sequencing uses
gaining all at a on genetic variation. Identification of spontaneous mutation
GWAS - what does it do
use of single nucleotide polymorphisms to link genetic variation to disease
GWAS uses
understanding pathways involved in complex disease. assessment of individuals disease risk
trio analysis - what does it do
identification of disease- causing spontaneous mutations
trio- analysis- uses
identification of the cause of rare genetic disease
knock out- what does it do
commonly in mice or model organism/ cell lines- disruption and inactivation of a specific target gene
knock out uses
assessment of gene function- creating disease models
CRISPR- what does it do
genome editing. allows user targeted disruption or alteration of mammalian genome
CRISPR uses
creating knockouts. therapeutic repair of mutant genomes
western blot what does it do
use of specific antibody to detect and quantify a particular protein in a sample
western blot uses
determine changes in protein live. Disease/ tissue specific expression
genetic screen- what does it do
random low level mutation of large numbers of a model organic,s
genetic screen uses
identification of genes important for a particular physiological or developmental process
ELISA- what does it do
use of specific antibodies or antigen to detect and quantify a particular protein or molecule in a sample
ELISA- uses
determine changes i protein level
-detection of viral protein, antibody responses and of secreted molecules
flow cytometry -what does it do
use of specific antibodies or fluorescent tags to detect proteins in intact cells
flow cytometry uses
identification and quantification of different cell types e.g. separation of cell types- sorting
immuno-fluorescence - what does it do
use of specific antibodies or fluorescent tags to detect proteins in intact cells using microscopy
immunofluorescence uses
visual determination of which cells express specific proteins. sub-cellular localisation of proteins.
