AMB6 Flashcards
(27 cards)
What is cDNA
complimentary DNA, copy of DNA synthesized from mRNA, via reverse transcriptase.
What is reverse transcriptase
an rna dependent dnap
What are the 7 steps to making a library
- extract rna
- 1st strand cdna synth
- 2nd strand cdna synth
- prepare ends
- clone into appropritae vector
- transform/package and infect
- library
What is the method for 1st strand cdna synthesis
Given 3’ poly A tail
primer used is poly T oligo primer
reversetranscriptase uses primer to make cdna complimentary strand.
what is the traditional method for 2nd strand cdna synthesis
- cdna:mrna hybrid denatured by boiling or hybridised with OH-
- hair pin loop formed at 3’ end by self hybridisation
klenow fragment e.coli is used as DNAP with dntps to form ds cDNA - remove hairpin with nuclease s1 non sequence specific digesting.
why is the traditional method for 2nd strand synthesis no longer used?
as the key step relies on the formation of a hairpin loop at end of first strand which forms by self hybridisation
what is the alternative method for 2nd cdna synthesis
- rnaseH cleaves rna strand leaving short pieces of mRNA hybridised to DNA
- remnants of mRNA serve as primers for synthesis of second strand cDNA
- Bacteriophage T4 DNA ligase sticks fragments together to form ds cDNA
- cap needs to be remoced and blunt ends treated
why is the alternative 2nd strand cdna synthesis better
as you lose none of the 5’end of the sequence so is more efficient
How are restriction sites inserted into cDNA ends
- use oligo primers with non complimentary homologous sequences including restriction sites eg. (T)nCATGCATG etc for both 1st and second strand
- seqyences can be extended using terminal transferase dnap indep of template - adds whatever base its gven.
- use different sequences either end to get assymetric ends that could be ligated.
originally how were cDNA ends made
polished using nucleases to produce blunt ends (no overhang) and then ligated to adaptors that produce ends with suitable overhangs for ligation vectors.
Describe the addition of 5’ or 3’ adaptors to cDNA ends (7) - cDNA directional cloning
- 5methyl dCTP used during RT of mRNA
- frist strand cDNA has xhol restriction site in primer
- xhol doesnt cut sites when contains methylated c
- rnase h and dnap used to make 2nd strand, then ligate adaptors.
- ecoR1 adaptors are ligated wihch is inefficient but driven to completeion by excess of adaptors
- end up with ecoR1 symmetric ends
- xho1 used to cleave site at is restriction site at primer end to make assymetric. methylation stops endogenous xho1 sites being cleaved.
how can the 5’ cap be exploited to get full length cDNA
- degraded unwanted rna doesnt have cap, so gives partial clones
- treatment with rna 5’ phosphatase removes P from 5’ of uncapped
- dephosphorylate uncapped and remove phosphatase
treat with tobacco acid phosphatase which hydrolyses 5’ cap end structure releasing an mRNA with only one phosphate group at 5’ end - only full length will be phosphorylated so only they can be ligated
- not1 restriction site with long re site is ligated to 5’’ end
then synthesize - get assymetric ends
why do all methods of cDNA synthesis get 3’ end of mRNA
due to primer
what is lambdazap?
artifical lambda phage that contains a plasmid surrounded by sequences recognised by a second type of bacteriophage - ensuring excision of plasmid/phagemid into second phage particles (happens in vivo in e.coli)
why is pBluescript a phagemid
as it contains an origin of replication f1 ( a virus) used to make ssDNA copies of plasmid.
what are the 4 stages of cDNA in vivo excision usuing helper phages
- lambdazap11 and ExAssist phages both infect E.coli
- if cell coinfected produce 3 types of phage lamdazap11, ExAssist and a new particle with ExAssist coat and pBluescript genome.
- Supernatant from infected E.coli used to infect different strains of bacteria (SOLR/XLOR) which have a genotype that doesnt allow infection by lambdazap11 but does ExAssist, but does not replicate ExAssist genome.
- some bacteria will therefore contain pBluescript that will replicate as a plasmid thanks to ColE1 ori
What are the benefits of of cDNA excision using helper phages
eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation.
Describe cDNA excision using Cre-lox
- very fast and require only single round of phage infection
lambda phage contains phagemid pTriplEx flanked by loxP sites
used to infect bacterium that expresses Cre-Recombinase enzyme
the two loz sites wil recombine to form an excised pTriplEx phagemid and an empty phage genome
Cre binds to lox site and cuts at both and strands are then rejoined by ligase
phagemid will replicate as a plasmid using ori
single Loxp site will remain as a molecular scar in plasmid.
why cant you talk about coverage in cDNA
as cdna is a copy of the mRNA and regularly expressed genes would have more RNA than less regularly express.
what is the usual size of a cDNA library?
usually cant get bigger than >4-5kb
what is representation in cDNA library?
simialr to coverage - wanted clones in libary.
why are rna probes more sensitive than dna probes for screen cdna lib
because rna:dna hybrid is reportedly stronger.
what can be used to screen cdna libs
heterologous clones eg. cDNA from common frog for rarer.
How can you get protein to cDNA and how can ambiguties be minimised
use mass spec and protein seq to do reverse translation and deduce possible coding sequences.
difficult due to degeneracy of genetic code = ambiguous
minimise this by choosing a target seq with fewer redundant codons eg. methionine and tryptophan only have one codon
also compare protein seqs to homologs of related proteins which sometimes share common domain blocks of conserved AA.
