Help: mutation screening in bacterial culture, SSCP? ddF?

Anders Gorm Pedersen gorm at cbs.dtu.dk
Wed Jun 7 04:26:29 EST 2000


(Crossposted to: bionet.molbio.methds-reagnts and bionet.microbiology.)

I am in the process of planning a project that will involve looking for
sequence variation (point mutations, and small insertions/deletions) in a
number of specific genes in E.coli. I have some ideas about how to proceed
but would be extremely greatful for any comments or suggestions from anyone
with experience in this type of work!

As part of the project I would like to look for sequence heterogeneity in a
growing culture of E.coli (e.g., a culture that has been propagated for many
generations by transferring a few ml overnight culture to a fresh 1 liter
shake flask daily). If at all possible I would like (semi)quantitative data
(i.e., "5% of this variant, 7% of that variant, 67% of that one," etc.).

My initial idea is the following:

(1) for each region of interest, construct PCR-primers that will amplify 300
bp (or so)
(2) isolate genomic DNA from an aliquot of the 1 liter flask (hopefully this
aliquot will be a representative sample of the entire culture).
(3) Run PCR on the genomic prep (perhaps using two differently labeled
primers, e.g., two fluorescent dyes)
(4) Analyze heterogeneity using SSCP (i.e., run labeled PCR product on
non-denaturing gel, each band corresponds to one sequence variant, the
intensity of the band is porportional to the amount of that variant).

I don't have (known) phenotypes for all the genes in which I'm interested,
so I don't think a genetic approach is feasible. (Plus, I want to catch as
many sequence variations as possible - including silent mutations).

Is this a reasonable approach?

I would like to be able to find even quite rare variations (say 1/1000), and
perhaps fluorescent labeling is not the best for this purpose - should I use
radioactivity?

The reason I considered fluorescent labeling, is that I have access to an
ABI-373A DNA sequencer. However, I do not have the ABI Genescan/genotyper
software (which can be used to size and quantitate DNA fragments). Should I
get this?

Do I have to isolate single colonies instead of looking at an entire
population at once? If sequence variations are present at low frequency this
will obviously be a LOT of work. (Of course I could isolate single colonies,
pool twenty or so, and screen the pools as described above?)

Is SSCP the best method? Should I use dideoxy footprinting (ddF).?  I have
the additional problem that the ABI-373A does not have temperature control,
so perhaps I will have trouble running non-denaturing gels? Are there other
good alternatives?

As mentioned any comments and suggestions would be greatly appreciated!

Thanks in advance,
Anders Gorm Pedersen

---------------------------------------------------------------
 Anders Gorm Pedersen, cand.scient., Ph.D.  (gorm at cbs.dtu.dk)

 Center for Biological Sequence Analysis
 Technical University of Denmark
 Bldg 208, DK-2800 Lyngby, Denmark

 phone: (+45) 45 25 24 84
 fax:   (+45) 45 93 15 85

 Web: http://www.cbs.dtu.dk/gorm/
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