cDNA Flashcards
Human genome size and % protein coding genes.
How many aa has a protein and number of bp
3x10^9bp, 1% protein coding (21000 gene - 3,2x10^7bp)
500aa, 1500bp
Capping of hnRNA, when and function
7-methylguanylate at 5´end in a 5-5´bond and 1st two bases are methylated - protects from degradation (pre-transcriptional)
Poly(A) tail where, when, function, how (describe process of polyadenylation)?
Poly (A) tail (~200 A´s) added at 3´end dowstream cleavage signal (AAUAAA) by PAP - stability of mRNA and helps transport to cytoplasm (post-transcriptional)
How poly(A) tail facilitates isolation of mRNA? how to isolate?
affitiny chromatography with primer 3´TTTTT (oligo dT chromatography) under high salt conditions, wash way other RNAs and elute mRNA (low salt)
Describe cDNA synthesis (Gubler and Hoffman)
First strand: oligo dT primer onto 3´poly A tail, reverse
transcriptase + dNTPs
Second strand: RNA H nicks mRNA (partial digest) creating
3´OH priming sites, DNA polymerase synthehesise strand
(5´-3´) using those sites and DNA ligase (T4) joins fragments
How can we improve ligation efficiency? Describe process
Adding 2 different adapters to make sure cDNAs are inserted in correct orientation.
Oligo dT at 3´end joined to an adapter NotI - first strand and then second
Adapter SalI to both ends
Digestion with NotI
One end as digested oligodT NotI and other SalI
Explain alternative way with 5´primer site and dsDNA adapter
Mn2+ and 2-4 cytosines and poly(A) with terminal transferase at tail 3´end
ligate dsDNA adapter complementar to tail
PCR with adapter primers or mRNA primer
4 library screening methods
in situ colony
in situ hybridization
oligonucleotide probes
antibodies
In situ colony (3 steps)
1- bacteria grow agar plate
2- colonies –> nylon filter (lysed - DNA/protein fix)
3- detection with DNA probe or Ab
In situ hybridization (5 steps)
1- blot colonies: nitrocelulose/nylon
2- lyse bacteria and fix DNA/protein
3- hybridise a probe to filter
4- wash undbound probe
5-auto radiograph
Oligonucleotide probes (4 steps)
1- select sequence and micro sequence (hexapeptide- small nº of combinations)
2-synthesise oligo, end label w/ 32P and hybridise with colonies
3- one oligo will have desired sequence
4- filter out by re-screening with probes based on another region of same protein
Antibodies (3 steps)
1- purify
2- raise polyclonal Ab and purify
3- probe colonies that have been lysed (proteins to nylon filter)
Original method (1970) and negative side
1st strand
alkali (remove mRNA)
loop to prime 2nd strand
S1 nuclease- remove loop
loss of information
Okayama and Berg method
plasmid primer and oligo dG linker (terminal transferase)
