Basic genetics in relation to DNA gene reg, gene expression and polymorphisms Flashcards
what does DNA finger printing rely on
variabel number of tandem repeats
myoglobin gene
segments in introns with no apparent function
- vary in size/composition between individuals
enzyme which cuts DNA
restriction enzyme
cuts at specific sequences
electrophoresis
run DNA fragments through gel
pass electric current accross
smaller the molecule faster it will run
what does gel electrophoresis separate fragments by
size
starting material for PCR
DNA
how do you reverse transcripbe to amplify
use RNA to make cDNA
polymerase chain reaction to make copies
polymerise chain reaction steps
open DNA by heating to 94 degrees, uncoils
add primers(forward and reverse), join at every end of DNA and corresponds
reduce temp to 54
extension occurs at 72 where new DNA is built
what is hybridisation
DNA sticks to DNA and RNA
RNA sticks to DNA and RNA
what can you do after amplification
run sequences on gel
show gene expression being associated with phenotype o the cell
problems with end point PCR
- poor precision
- lower sensitivity
- short dynamic range
- low resolution
- non automated
- size based discrimination only
- results not expressed as numbers
- ethidium bromide staining is not very quantitive (likes to stick to nuclear material)
what does real time PCR detect
accumulation of amplicon during reaction
what is the optimal point for analysing data in real time RCR
exponential phase
why can you not take data at the plateau in real time PCR
different amounts of DNA in the starting material
- data would not truly represent starting materail
what are the methods for real time PR
Taq man probe
flurorences
Taq man probe method
has a quenching and reporter dye
- Taq man probe and primer binds to target DNA
- Taq polymerase can now create complementary strand
- Reported dye released from the extending double stranded DNA
- reporter dye now distant to quencher so it will fluoresce and give a signal
- machine analyses this
what does light relate to in taq man probe method
amount of light relates to amount of DNA in sample
2nd method for real time PCR
- cyber green attatches to any double stranded DNA
- When the DNA is denatured the green floats free
- can then pick up the fluorescent activity by the machine
advantages of real time PCD
traditional measure at end point and real time collects date in exponential growth phase
increase in reported fluorescence directly proportional to number of amplicons generated
increased dynamic range of detection
no post PCR processing
transcriptomics
genome wide RNA transcript expression levesl
in situ hydribdication
localisation technique for the detection of a gene product (RNA) in tissues
what can in situ hybridisation be used for
investigate cell type and tissue specific gene expression
genomics includes..
recombinant DNA
DNA sequencing methods
bioinformatics
- used to sequence, assemble and analyse the function/strucute of the genme
proteomics
large scale study of proteis
metabolomics
comprehensive characterisation of small molecule metabolites in biological systems
what is a nucleotide polymorphsm
single nucleotide change in genome sequence
some no effect, some lead to disease
genome wide associated studies
rapid scanning of complete sets of DNA of many people to find genetic variations associated with a particular disease
Taq man probe method
has a quenching and reporter dye
- Taq man probe and primer binds to target DNA
- Taq polymerase can now create complementary strand
- Reported dye released from the extending double stranded DNA
- reporter dye now distant to quencher so it will fluoresce and give a signal
- machine analyses this
what does light relate to in taq man probe method
amount of light relates to amount of DNA in sample
2nd method for real time PCR
- cyber green attatches to any double stranded DNA
- When the DNA is denatured the green floats free
- can then pick up the fluorescent activity by the machine
advantages of real time PCD
traditional measure at end point and real time collects date in exponential growth phase
increase in reported fluorescence directly proportional to number of amplicons generated
increased dynamic range of detection
no post PCR processing
transcriptomics
genome wide RNA transcript expression levesl
in situ hydribdication
localisation technique for the detection of a gene product (RNA) in tissues
what can in situ hybridisation be used for
investigate cell type and tissue specific gene expression
genomics includes..
recombinant DNA
DNA sequencing methods
bioinformatics
- used to sequence, assemble and analyse the function/strucute of the genme
proteomics
large scale study of proteis
metabolomics
comprehensive characterisation of small molecule metabolites in biological systems
what is a nucleotide polymorphsm
single nucleotide change in genome sequence
some no effect, some lead to disease
genome wide associated studies
rapid scanning of complete sets of DNA of many people to find genetic variations associated with a particular disease
