Lecture 5: DNA and RNA manipulation Flashcards
what are the 3 diff kinds of blotting, and what are they for
- southern DNA-DNA
- northern DNA-RNA
- western antibody-protein
what does a western blot detect
used to detect or idnetify specific proteins in homogenized tissue
describe how southern blot works
- a restriction enzyme is added to a solution containing the DNA of interest
- the resulting fragments are separated by size by gel electrophoresis
- after electrophoresis, the DNA fragment sin the gel are denatured and then transferred (blotted) onto filler paper
- after blotting, the paper is removed
- the paper is placed into a plastic bag w a solution w the single-stranded, labeled probe, the probe sticks to complementary frgaments by hybridization
- to visualize the positions of the bands where the probe is hybridized, the filter paper is exposed to x-ray film, and either light or radioacative emissions darken the film over each band
are DNA fragment bands visible during step 5 of southern blot
- no
- they need to be exposed to x-ray film and either light or radioactive emissions
how does northern blot work
- Harvest total RNA
- Separated by agarose gel electrophoresis
- Hybridize a labelled probe to the blot
- Detect as before
- More RNA the band contains, the more the probe it will bind, and the darker the band will be on the film
the more RNA the band contains in northern blllot, the ____ (lighter/darker) the band will be on the film
darker (cause itll bind to more probe)
what are the target molecules in southern, northern, and western blot
- southern DNA
- northern RNA
- western protein
how do you prep the samples in southern, northern, and western blot
- southern DNA extraction, enzymatic digestion
- northern RNA isolation
- western proteine extraction
what are the separation methods for southern, northern, and western blot
- southern electrophoresis
- northern electrophoresis
- western electrophoresis
what is the membrane material in southern, northern, and western blot
- southern nylon
- northern nylon
- western nitrocellulose or PVDF
what probes are used in southern, northern, and western blot
- southern nucleic acid probe w sequence homologous to target
- northern RNA, DNA, or oligodeoxynucleotide
- western primary antibody
what is the probe label in southern, northern, and western blot
- southern radiolabel, enzyme
- northern radiolabel, enzyme
- western enzyme
what are the detection methods in southern, northern, and western blot
- southern x-ray film, chemiluminescence
- northern x-ray film, chemiluminescence
- western film, cooled CCD, camera, LED or infrared imaging system
true/false southern, northern, and western blot all do separation by electrophoresis
true
true/false southern, northern, and western blot all use x-ray film and chemiluminescence as a method of detection
- false
- southern and northern do
- western uses film, cooled CCD, camera, LED or infrared imaging system
which 2 blot techniques have the same membrane material
- southern and northern
- nylon
what are 2 ways to label DNA
- direct
- indirect
true/false DNA interference is a defense mechanism
true
what is the main purpose of RNA interference
to regulate gene expression
What are the potential fates upon RNA interference in eukaryotes
- Rapid cleavage of target DNA
- Rapid translational repression, and (typically) eventual destruction of target RNA
- Formation of heterochromatin on DNA from which target RNA is being transcribed
describe how RNA interference works
- Begins w the introduction of long Double stranded RNA (dsRNA). This can be natural (viral infections), or artificial (labs). it is formed vis complementary base pairing w itself
- This RNA is “cropped” while still in the nucleus and then exported into the cytosol
- in the cytosol, the enzyme “dicer” recognizes the dsRNA and cleaves it into short 20-25 nucleotide long fragments called siRNAs, or miRNA proper
- Argonaute, along with other components of RISC complex, associate with both strands of miRNA and then cleaves and discards one of them (the passenger strand)
- The other strand (guide strand) is loaded into the RISC complex
- RISC contains helices that unwind the siRNA, which can now bind to complementary mRNA sequences
- The guide strand binds to a complementary region on the target mRNA through base pairing, forming an RNA duplex
- If the RNA-RNA match is extensive, aeronaut cleaves the target mRNA causing rapid degradation
- If the RNA-RNA match is not longer than 7-nucleotide regions rapid inhibition of translation and eventual destruction will occur
true/false in many organisms, the double-stranded RNA can trigger both the destruction of complementary mRNAs and transcriptional silencing
true
can RNA interference direct heterochromatin formation
yes
how can RNA interference direct heterochromatin formation
- The short siRNAs produced by the dicer protein are assembled with a group of proteins to form a RITS (RNA-induced transcriptional silencing) complex.
- This will bind complementary to RNA transcriptis as they emerge from a transcribing RNA polymerase
- The RITS complex will attract enzymes that covalently modify nearby histones and DNA causing the formation of a “constitutive” form of heterochromatin
- This will self propagate and prevent RNA from being transcribed
true/false RNA interference is not a good way to test RNA function
- False
- RNA interference is a simple and rapid way to test gene function
what are the steps of CRISPR
1) Short Viral DNA sequence is integrated into CRISPR locus
2) RNS is transcribed from CRISPR locus, processed and bound to Cas protein
3) Small cRNA in complex with Cas seeks out and destroys viral sequences
what is CRISPR used for
gene editing
what does CRISPR simplify
making transgenic organisms
