Strategy Questions
Remember, clearly define the problem you want to solve or specific question you want to answer. Then ask yourself: 1)
What do I need in order to do the experiment, produce the product, etc? and
2) How do I go about finding/constructing?
I.
Finding/Isolating the gene or cDNA you want to work with
Cloning in E. coli
Do I want a cDNA or a genomic library? If I want a cDNA, can I circumvent making a whole library by partially
purifying the mRNA of interest?
What vector should I use? [plasmid, lambda (insertion or replacement), cosmid]
How will I make my construct?
How will I get it into E. coli?
How will I recognize successful transfer into E. coli?
Can I tell vector onlies from vector+inserts?
How will I find which clones have the right insert?
A.
Probe colony or plaque filter lifts
1.
Nucleic acid hybridization to DNA on filter
a.
Previously isolated clone which shares sequence homology?
b.
Synthetic oligonucleotide based on amino acid sequence?
c.
Previously known marker probe to start a walk or jump?
i.
Additional tests to identify which clone along the walk is the one of interest
2.
Antibody binding to protein product on filter (requires an expression vector)
B.
For a subclone or a clone with a known map
Isolate the DNA, perform RE digestion and gel electrophoresis, and compare to a known map.
Cloning Enormous Pieces of DNA
If you want to clone an enormous piece of DNA (>=40kb)
1.
Use E. coli with
a.
BAC, bacterial artificial chromosome, based on F plasmid
Takes inserts up to 300 kb in size
b.
Bacteriophage P1. Can handles inserts from 75-100 kb.
2.
Use yeast with a YAC vector, yeast artificial chromosome.
Depending on type of YAC vector, inserts are 600-1400 kb in length.
Lift yeast colonies onto filters and probe for specific gene using nucleic acid hybridization.
II.
Using the gene you have isolated for further studies
A.
Structure studies - may require subcloning into an appropriate E. coli vector
Restriction mapping
Sequencing
B.
Function studies, knockdown studies, knockout studies, or expression of the gene product for market or
further study.
To do these experiments you need to design your construct with care, paying attention to what features
are required in both the vector and the insert. Remember, once you've designed and made your
construct in E. coli, you grow a sufficient amount in E. coli before you transfer it into another cell type.
You need to ask yourself:
1.
What recipient cell type do I want to use?
2.
Should I express from a promoter-insert construct or a genomic construct with its own promoter? Do I
have an acceptable promoter and regulatory commands for the recipient cell of interest?
3.
Do I want stable or transient expression?
4.
If stable, do I want the gene integrated into the host cell genome or independently replicating?
a.
If integrated, is random insertion OK or should I select for homologous recombination (gene
targeting)?
i.
b.
If randomly inserted, do I want predictable flanking sequences such as LTR or border
regions?
If independently replicating, do I want high or low copy number? (Particularly relevant in yeast)
5. How will I introduce the construct into recipient cells?
Remember, if you choose pseudoretroviral transfer, you need to include an extra gene transfer step just
for the purpose of packaging.
6. If stable, how will I tell which recipient cells have successfully taken up the DNA?
a.
Selectable marker
b.
Some other assay
7. How will I test for expression or loss of expression of the gene of interest?
Notes
1.
2.
If you want to make mutations to test their effect on expression or function, mutate your construct in the test tube.
Transform into E. coli and grow up enough mutant DNA in E. coli to use for gene transfer.
If you want to test promoter sequences for efficiency, consider a transient assay using a vector with a reporter
gene such as CAT and a cloning site into which you can insert the fragment containing the promoter sequence.
Have fun reviewing and do well on the exam!