lecture 5- methods & microscopy Flashcards
what is pronuclear microinjection
DNA is injected in the embryo and taken up into the genome
- initial problem: “positional effect,” DNA integration was random, so different transgenic often had different expression due to their position in the genome
- homologous recombination
what is homologous recombination and what became the problem & solution
- targeting of inserted DNA to a specific site in the genome
- through the exchange of equivalent amount DNA from organism and foreign plasmid
- allows one to remove an entire gene, “knockouts”
- insertion of another gene “knock in” –> lethality became problem
solution was conditional knockouts via the CRE-LOX system
describe homologous recombination steps
identify gene u want to knockout (YFG)
- generate a plasmid or other DNA vehicle that contains an empty region that is flanked by border sequence identical to YFG
–> put plasmid in with genomic DNA often in embryo –> matching flank sequences will align & recombination will occur
–> now u have targeted and eliminated YFG from your genome
describe conditional/inducible homologous recombination using CRE-LOX
lox sites are 34b of sequence from bacteria phage, these can be placed into a genome (flank a gene)
- CRE is a specific site recombinase that excises material in between LOX sites
in homologous recombination with CRE-LOX, f you express CRE with a cell or tissue or now an inducible promoter, then…
then you can cause a knockout in a specific cell or tissue under your control
for homologous recombination, why might conditional or inducible be better?
you control the effects
- temporally- if a gene if required in the embryo, then you can now see what it is doing later on in development, by not knocking it out until after the embryo is formed
- specific conditions- certain stresses or after infections
spatially- what happens in one part of the organism
name advantage and limitation of CRISPR
advantage- targeted gene editing
limitation- still requires transformation; off site (extra) targeting sites
new: Cas13 - RNA editing and Cpf1 - different cut and editing sites
name 5 methods of transcript analysis (RNA expression analysis)
1- northern blot
2- RT-PCR
3- Realtime PCR (qPCR or qRT-PCR)
4- microarray
5- RNA-seq
describe method of northern blot
RNA is isolated, run out on a gel to give a size gradient spread and transferred to a nylon membrane –> DNA of choice is used as the probe (radioactive) that will anneal to complementary RNA
in northern blot, the amount of signal = __
the amount of RNA or gene message present
in northern blot, running a gel (electrophoresis), it separates by ___ using ___
size
charge
PCR-based RNA analysis methods
reverse transcriptase (RT-PCR) vs. real-time or quantitative PCR (qRT-PCR)
- both PCR based methods to determine gene expression levels in samples
- RNA is converted back to DNA (cDNA) on which PCR can be conducted
- amount of PCR product depends on original amount of RNA present (transcript or gene signal)
PCR is a method to ___/___ DNA
copy
amplify
describe RT-PCR (reverse transcriptase)
extracted RNA is converted back to DNA (cDNA) on which PCR can be done in a semi-quantitative manner
- amt of PCR product depends on original amt of RNA present (** this can only be observed by ending the PCR rxn**)
describe real time (qRT-PCR or qPCR)
a fluorescent dye or labeled primer is added to the PCR rxn of cDNA from a RNA sample and then a laser monitors the amt of fluorescence made at every PCR cycle
in realtime (qRT-PCR or qPCR), fluorescence corresponds to ___
excellent method for…
the amt or expression levels of RNA (gene signal)
excellent for examining extremely low RNA transcripts (signals)
describe microarray
- like a scaled up northern using fluorescent dyes
- except use “blotted” DNA with RNA as the probe
- DNA (in parts) if often most of the entire genome
in microarray, you isolate ___ and label with ___ and probe all ___
RNA
fluorescent dye
genes
in microarray, the amount of fluorescent signal =
amount of gene expression under RNA isolation conditions
microrrsay vs. RNA sequencing
microarray: relative intensity = expression levels
RNA sequencing: sequencing reads = expression levels
describe RNA sequencing
transcriptome-wide expression analysis
get mRNA –> convert to cDNA and cut –> add labeled ends –> use PCR to amplify small fragments of cDNA –> perform next generation sequencing –> assemble transcripts
RNA sequencing is great for…
detection of very low copy transcripts
also for whole genome based changes (sequenced genome not required)
quantitation of specific transcript levels between samples
name of the standard light microscope
compound
light microscopy with polarizing lens
DIC (differential interference contrast)
