mRNA Flashcards
describe 5’ race
gene-specific primer, synthesize cDNA from the mRNA strand towards the 5’ end (on the mRNA); using terminal transferase, add on a polyA tail to the 3’ end of the cDNA; using a polyT primer and DNA pol, synthesize the other strand; amplify DNA using adaptor primers 1 and 2; cleave adapter-primer –> Done!
issues with 5’ RACE?
many partial cDNAs–>lots of fragments of various lengths, makes it difficultt o get to the 5’ end of the full-length cDNA;
what is 5’ anchor ligation?
improves method of obtaining full length cDNAs; if a cDNA is full length, it will have the 7-meGuanosine cap; therefore, when alkaline phosphatase comes around and dephos’s 5’ phosphate, it can only do it on the (partial) cDNAs with no cap–> when adapters are added (after removing CAP With tobacco acid pyrophosphatase) they cannot bind without a 5’ phos–>no partial cDNAs which can be amplified
describe northern blot procedure
isolate mRNA via polyT oligo chromatography; resolve on gel and tranfer to membrane; hybridize to labelled DNA/RNA probe-->P32 or S35 nts or biotin/dioxigenin-labelled nts detect via autoradiography--x ray film or enzyme (alk phos)-->colourmetric, luminescence-->Abs!!!
advantages and disadvantages of northern blot?
• Advantages – Size determination of mRNA species – Blots can be “stripped” and re-probed – Simultaneous detection of mRNA species in different tissues • Disadvantages – Detection with one probe at a time – Time consuming, low throughput – Not very sensitive (relatively to newer methods); requires more RNAs
describe in situ hybridization
• Preparation of tissue sections onslides
– Fix in formalin and embed samples in paraffin – Thin sections are mounted on slides
• Hybridization reaction
– De-wax and re-hydration
– Proteinase K digestion to improve access of probe – Hybridization with labeled probe
• Detection
–use antisense probe on sense strand, sense probe for control
in situ hybridization adv and disadv?
• Advantage --localize of mRNA in tissues • Disadvantages – Labor-intensive procedures – Quantification is difficult – Not very sensitive – No information on sizes of mRNAs
describe traditional RT-PCR
• cDNAsynthesisbyreversetranscriptase
• PCRamplificationwithtarget-specificprimers
• Detection by gel electrophoresis or fluorescence-based methods
Quantification
- compare the amount of product in a gel
- use of internal controls: amplification of stably expressed gene transcripts as loading controls
- not precise, considered “semi-quantitative”
what problems are inherent in RT-PCR?
– PCR is an exponential process
→ small differences in efficiency at each cycle can lead to large differences in the yield of the amplified product
– Example:
traces of inhibitors in different samples affect efficiency in primer-template annealing
describe qPCR
real-time quantitative PCR–method to measure the amount of template you put in
• Monitorfluorescencesignal(DNAsynthesis)inreal time
• TypicalamplificationplotandCT
– Three phases and baseline
– CT : threshold cycle at which PCR product signal is detected
• There is a linear relationship between CT and the logarithm of the initial template DNA concentration
• Astandardcurvecanbeestablishedandusedto calculate the concentration of a target gene
how to generate fluorescence signal in qPCR?
(1) Intercalating dyes - SYBR green
(2) Probe-based technologies
- Taqman: hybridize and hydrolyzed
- Molecular beacon: hybridize but not hydrolyzed
(3) Primer-based technologies - fluorogenic LUX primer
SYBR green–intercalates into DNA bases, also minor groove; fluorescence doubles when between dsDNA; however, it is non specific
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describe the realtime PCR signal Taqman?
taqman probe has reporter (fluorescent) and quencher; when DNA is synth’d the bound probe (which is on a specific segment of DNA) is displaced and the R is separated from the Q; now the R can give off fluor; the more template, the more the R given off–>more fluor!
what is a molecular beacon?
hairpin probes with R and Q bound to each other; during the annealing stage, probe binds to ssDNA and R fluors
describe the fluorgenic LUX primer
hairpin R with no Q; just being in a hp quenches it (Guanosines in close vicinity); when it anneals to DNA and is extend is fluors
