Lecture 3 Flashcards
Bacteriophage undergo either….
lytic replication or lysogeny once it has infected E.coli
What happens in lytic replication?
ACTS AS PLASMID- genome not incorporated
1) Transcription and translation of early genes that help to replicate viral DNA
2) Huge replication of phage DNA
3) Then get expression of late genes that encode head and tail region
4) Self assembly of fully active new phage particles that lyse open bacterial cell and spread particles
Lytic replication of Bacteriophage results in cell death of E.coli and formation of ….
clear plaques on cell lawn
How do you plate Bacteriophage ?
1) Plate is covered in lawn of bacteria that act as host cells
2) Add dilute suspension containing virus, after infection cover layer of cells with agar and incubate
3) Each plaque represents cell lysis initiated by one viral particle (agar restricts movement so that virus can only infect contiguous cells)
How is the DNA in a bacteriophage replicated?
Enzymes for replication go round circle and produce long molecule of genome joined together= concatemer.
- Endonuclease A recognises cos sites and cuts at them
- Results in short pieces of DNA that contain viruses entire genome
- It is this that gets packaged into phage particles
What is meant by the fact bacteriophages are self assembling?
The addition of DNA containing COS sites at the correct spacing (46-53kb) allows spontaneous formation of complete functional phage.
(Basis of in vitro packaging system)
What was wrong bacteriophages?
- Upper limit between cos sites only allows for 3.5kb to insert DNA into
- Not useful as does not giver larger capacity than plasmids (can add 20kb to them)
How did they make phage vectors better?
- phage vectors have a central stuffer fragment which is replaced by DNA to be cloned (DNA removed is for lysogenic pathway so not needed)
- As long as replaced with DNA of equal size
- CAN NOW add 25kb of DNA into system
- NO determent to transfection efficiency when inserting large DNA.
When would you use a phage vector ?
If you wanted to clone DNA over 20kb (up tp 25kb) as transfection efficiency constant over size range
Why are plasmids not a good cloning vector for large DNA?
Transformation falls off rapidly with insert size
Which clones are easier to handle, bacterial clones or phage clones?
Bacterial
Plating density of phage can be how high?
> 10,000 per plate
(Good if you want to screen for particular gene- easier to find clone interested in)
GOOD for large libraries
TRUE or FALSE
You can generate a nearly complete genomic library of higher organisms by phage cloning?
TRUE.
How do you package the DNA to be replicated into a phage viron?
1)Partial digestion with Sau3A into 20-kb fragments
2)Remove stuffer fragment from bacteriophage with BamHI
(Sau3a and BamHI both create overhanging ends)
3) Mix human DNA fragment and phage arms - seal with DNA ligase
4) Recombinant phage DNA packaged with in vitro phage-assembly system
=Recombinant phage virion containing human genomic DNA
Why is it NEAR complete genomic library?
Only digested human DNA using one restriction enzyme so can be difficult to put together.
Better to degrade DNA with different restriction enzymes to get overlapping fragments.
Even better use mechanical sheering for random breaking down of DNA.
cDNA libraries are prepared from..
isolated mRNAS
How do you get mRNA?
- RNA extraction, treated with a DNAse but is still a mixture of cytoplasmic RNAs not just mRNA
- Need to use hybridisation to stable matrix to get just the mRNA
- 5’ polyadenylated tail of mRNA binds to T’s on matrix
- Wash away contaminating RNAS e.g. tRNA and elute mRNA molecules
How do you produce cDNA from mRNA?
1)Reverse Transcriptase makes DNA copy from RNA template (needs primer)
2)Add oligo-dT primer
3)Reverse transcriptase + nucelotides makes new chain
4)RnaseH recognises this DNA/RNA hybrid and degrades RNA molecule
5)Then add DNA polymerase to make a DNA copy of RNA molecule
(Can add linkers to end of DNA to help join to vector)
Preparation of a bacteriophage cDNA library
1) Introduce EcoR1 methylase to protect cDNA from restriction enzymes
2) Ligate cDNA to ECOR1 linker
3) Cleave with EcoR1
4) Ligate to phage arms, package in vitro, infect E.coli
Features of genomic libraries
- Cloned DNA directly from genome
- Generally only library needed for prokaryotes
- Need to contain more clones to present entire genome
- Cloned fragments contain introns and exons
Features of cDNA libraries?
-Cloned DNA fragments are copies of mRNA sequences
-Cloned fragments contain only transcribed regions
-Contain only exons
-Libraries made from different tissues and/or developmental stages of organism differ because different genes transcribed into mRNA
(Gives idea of whats occurring NOW in organism)
What can you screen a library for?
Enzyme activity
Protein the gene encodes (detection based on gene product not always practical as need to know a lot about protein to begin with)
How is screening done on the basis of activity?
Example: cloning amylase gene
- Amylase degrades starch
- Plate out library on starch-containing agar
- Flood plate with Iodine solution
- Clone containing amylae gene has a halo where starch digestion takes place
How is screening with antibodies done?
- Antibodies used to detect a specific epitope of protein in a complex mix and amount of protein
- Plate out library on agar
- Transfer to filter
- Lyse cells to release protein
- Add inert protein to block non-specific binding
- Incubate with radioactive antibody
- Expose to X-ray film and detect where antibody binds
