Making DNA libraries via DNA cloning Flashcards
What is DNA cloning for?
What are the 3 elements required?
What is a DNA library?
Isolate gene of interest- characterise & manipulate it
cDNA, reference source/DNA library & vector
Storage of DNA fragments stored in host bacterium & used as point of reference for searches- can be genomic or cDNA
How do you obtain genomic DNA fragments from a DNA library source?
What about cDNA fragments?
What do you do with the obtained DNA?
What’s the difference between the genomic DNA & cDNA?
Restrict with nuclease digestion.
Transcribe the DNA into RNA transcripts, which are then spliced (remove introns) into mRNAs & then reverse transcripted.
Restrict & clone into vectors.
Genomic = promotor, regulatory elements & introns cDNA = material from mature mRNA so only exons
What are the 4 steps from DNA to mRNA?
What is the PolyA tail used for?
What are the 4 steps of this process?
- RNA polymerase II- transcription & capping of 5’
- Cleave poly(A) site with endonuclease
- polyadenylation with poly(a) polymerase & ATP adding adenosine 100-250 at 3’ end
- Splicing- end up with mRNA with A100-250 on 3’
Separating mRNA from RNA mixture
- Chromatography column w/ cellulose matrix with cov linked oligo chains- add solution RNA in 0.5M NaCl
- Wash with 0.5M NaCl remove unbound RNAs & pass through column (mRNA with poly A tails which hybridise to oligo on cellulose)
- Elute mRNA from column with H2O
- Collect & evaluate
What happens once you add reverse transcriptase to mRNA?
How can you separate this structure? How do you produce a double stranded cDNA from this?
How do you degrade the mRNA once separated?
Why is cDNA better?
End up with DNA/RNA heteroduplex
Separate with heat- DNA polymerase
Orinase
More stable storage of only actively transcribed genes in sample
What are the 2 methods for a DNA polymerase primer to produce cDNA if you don’t know the target sequence?
What are their limitations?
What’s the best method?
- Oligo dT priming-primes (at poly A tail of mRNA)
- random priming-random (hexamers of oligonucleotides for all possible combinations)
Oligo- only eukaryotic DNA & can skew cDNA towards 3’ of gene
Hexamers- 4096 possibilities
Use both
What is a cDNA library?
How do you prepare the cDNA before cloning into the plasmid vector?
How is this then inserted into the plasmid vector?
What does this form?
In what region of the plasmid is the DNA inserted?
What does the ORI region on the plasmid allow?
Stable repository of all actively transcribed genes in source mRNA- can be tissue specific to narrow search
Cleave with type 2 restriction endonuclease to make sticky ends (cleaves fragments between restriction sites)
Restrict the vector with the same enzyme to produce compatible ends- then anneal the sticky ends together & ligate with DNA ligase
Recombinant DNA molecule
Polylinker (where contains DNA for numerous restriction enzymes)
Replicate in the host bacteria
How does transformation work in isolate the competent bacteria (with the recombinant DNA/plasmid)?
What do you do with these colonies?
How could you identify a clone of interest?
What are the steps involved?
What probes could you use?
Plate bacteria on agar plate with E.coli- the only colonies left will have taken up the recombinant plasmid.
Choose one and incubate in growth medium & isolate plasmids from bacteria & purify
Colony screening by hybridisation
- Place colonies on nitrocellulose filter & incubate in alkali solution to lyse cells & release plasmid single strand DNA.
- Hybridise with labelled DNA (probe) & wash away unhybridised DNA.
- Perform autoradiography- signal appears where DNA is complementary to probe.
- Related gene sequence (homologue)
- previously cloned part of gene
- cDNA from other tissues (label when reverse transcribe mRNA)
- reverse engineered probe from protein of interest (from oligonucleotide probes)
