DNA and RNA analysis Flashcards
what are several methods of analysing DNA?
- DNA visiualisaiton; agarose gel electrophoresis
- Amplification; PCR
- Sequencing; Sanger and Next Generation
what is karyotyping?
the process of preparing chromosomes for analysis.
what is a karyotype?
number and appearance of chromosomes in the nucleus of a eukaryotic cell.
what does chromatin look like under analysis?
heterochromatin; dark > inactive
euchromatin; light > active
what is FISH?
fluorescence insitu hybridisation
- staining technique where known sequences of DNA are fluorescently labelled and hybridised to chromosomes
what does in situ refer to?
the fact that the fluorescent DNA probe is hybridised to DNA of interphase nuclei or metaphase chromosomes which have been fixed on a slide
what does FISH provide researchers with?
a way to visualise and map the genetic material in an individuals cells, including specific genes or portions of genes.
what can FISH only do?
detect deletions or duplications of regions specifically targetted by the probe used.
PCR
- exponential amplification of a DNA fragment
- usually of known sequence
what are the components used in PCR?
- template; DNA to amplify
- primers; short pieces of ssDNA
- polymerase; thermostable enzyme (Taq)
- nucleotides; single base mixture (dNTPs)
- buffer; to maintain pH
- MgCl2; essential for polymerase activity.
principles of PCR
- requires incubating at three different temperatures
= denaturing, annealing, extension
background of Sanger sequencing
- invented by Frederic Sanger and co in 1977
- also called dideoxy sequencing because it modified nucleotides called dideoxynucleotides
what is process of Sanger sequencing?
- DNA extracted from the cells of the organism being studied
- sequencing reaction the performed on the DNA and the sequenced DNA strands are sorted by size using capillary electrophoresis.
- finally DNA code is read by a computer which displays the data for scientists to use.
summary of what sanger sequencing is
A PCR containing fluorescent, chain terminating dideoxynucleotides triphosphates
why are fluorescent in sanger sequencing called chain terminating?
because they contain an alteration in the structure that doesn’t allow polymerase to continue sequencing.
- ddTTP, ddATP, ddGTP, ddCTP
what are the main causes of cellular dysfunction that underlies different disease states?
disturbances in gene expression as a result of pertrubed transcription or posttranscriptional regulation
what is qRT-PCR used for?
for gene expression analysis
- generate cDNA from mRNA, then PCR
what is microarray used for?
detect the expression of thousands of genes at the same time
what is RNA-seq used for?
allows for full sequencing of the whole transcriptome
central dogma of molec. biology
DNA > RNA > protein
what is reverse transcriptase-PCR?
generation of cDNA from RNA that will be amplified and run on agarose gel to look at expression of gene of interest in different tissues
- assesses transcript expression
what is a housekeeping gene?
gene that’s expression will not be affected by anything done to the tissue or cell.
steps of qPCR
1st step; reverse transcription (RT)
2nd step; PCR using SYBR green fluorescent; can bind to ds DNA , unbound will not emit fluorescence
3rd step; fluorescence measured
how are results taken in from qPCR?
- output measured in real time in the thermal cycler
- number of cycles taken to reach the threshold value is determined
- proportional to the amount of starting cDNA (mRNA)
summary of DNA and RNA analysis
- we can visualise and separate DNA fragments based on the size with the agarose gel electrophoresis
- karyotyping and FISH can visualise and map genetic material and detect gene deletion or duplications
- PCR is used for DNA amplification and for the Sanger/Dideoxy sequencing
- can quantify differences in mRNA expression with qPCR. If a particular seq is abundant in the sample, amplification if observed in earlier cycles; if sequence is scarce, ampification observed in later cycles.
