Q: purified primers for sequencing??????
Dr. Michael T. MacDonell
sendero at ix.netcom.com
Sun Apr 27 10:50:44 EST 1997
In article <Pine.GSO.3.95.970426155856.7374A-100000 at dale.ucdavis.edu>,
Mysore Sudarshana <szsudhi at mailbox.ucdavis.edu> wrote:
>On Tue, 22 Apr 1997, Dr. Duncan Clark wrote:
>> In article <5jhjih$el2 at light.nih.gov>, Bernard Murray
>> <bernard at elsie.nci.nih.gov> writes
>> >Maybe you can use very crude oligos for some applications
>> >(I've never done it) but gel purified oligos have been fine for me for
>> >PCR (Taq) and sequencing (Sequenase). Some methodologies may require
>> >the full HPLC purification.
>> We have an old 381A synthesiser and I use crude oligos for everything
>> from PCR to sequencing to long PCR of 20kb. By crude I mean deprotected
>> and just ethanol pptd. Running them on an FPLC every so often shows them
>> to be pretty clean. Using purified compared to unpurified gave no
>> difference for the PCR reactions we tested them on.
>> The problem with being on the cutting edge is that you occasionally get
>> sliced from time to time....
>> Duncan Clark
>> DNAmp Ltd.
>> TEl/FAX 01252376288
>I am not an expert on primer synthesis. The primers (unpurified or simply
>undesalted) come with lot of salts which can be a pain in automated
>sequencing or in site-directed mutagenesis. We get pur primers
>synthesized in central facility at UC-Davis. The cheapest primer comes
>with trityl group removed and lyophilized. For desalting we have to pay
>extra. This we use directly for sequencing with sequenase kit (in our lab)
>or using ABI sequencer at a central facility.
>If the primer is not gel purified, it can be expected to have a very small
>proportion of shorter chains. The coupling efficiency is pretty high these
>days. The shorter chains correspond to a very small proportion and it is
>unlikely to pose a problem in reading the gels.
>I am quoting a reference for purifying deprotected oligodeoxynucleotides
>wherein the authors have used n-butanol and the purification takes about
> Sawadogo, M. and Van Dyke, M.W. 1991. A rapid method for the
> purification of deprotected oligodeoxynucleotides. Nucleic Acids
> Research, 19: 674.
>I have used this method previously, when the oligo came as a solution in
>ammonium hydroxide. Of course, that time I was doing sequencing manually
>using sequenase 2.0 kit.
>Dept. of Plant Pathology
>Univ. of California,
>Davis, CA 95616.
There has been an unfortunate misuse of the term
"purified" when applied to the quality of synthetic
oligonucleotides. The misuse derives from some
less-than-scrupulous marketing practices among the
various suppliers. Whatever we mean to convey when
we say "purified" shouldn't be "desalted", other-
wise it becomes difficult to justify the existence
of two terms used to define the same thing.
Gel purified, regardless of what it means to convey
to the imagination of the user, actually means
"desalted". Just as "desalted" means "desalted".
Desalting can be accomplished by a variety of means
and gel filtration is one of them. Likewise,
another misleading term is "column desalted" which
is honest enough as to the fact that desalting is
taking place, but avoids the distinction whether
the desalting is done on a reverse-phase column
(expensive), or a Sephadex column (somewhat less
expensive). I have little doubt that the origin of
the term "gel purified" is rooted in the hope of
some users mistaking it for PAGE purified. Indeed,
there is a world of difference. Whereas nothing
renders a purer oligo than PAGE purification,
gel purified only desalts.
Possibly a good starting place is to define what
is meant by purification, when applied to oligos.
Since nothing can be done about the small freq-
uency of spurious chemical modifications that
occur, impacting both the individual nucleotide
bases and the phosphate backbone, purification
must address length homogeneity, i.e. elimination
of all of the "failed" sequences or fragments that
are a consequence of the less-than-quantitative
nature of the coupling reaction. OPC purification
(also called trityl-specific purification) is
probably the minimum treatment that can qualify
for "purification". HPLC is better, but is vastly
more expensive and delivers very minimal yields.
PAGE purification, actually electrophoresing (is
that even a word?) the oligo through a poly-
acrylamide gel, is the best you can aspire to.
However, PAGE purification is not only expensive
and delivers minimal yields, but it is (like HPLC)
usually not needed for most applications.
So, what is actually needed? For PCR, crude oligos
will do. Many investigators with DNA synthesizers
in their labs, just deprotect and use oligos for
PCR without any other fuss. Ethanol precipitation,
which virtually - though not completely - desalts
oligos is better, and can also be used for DNA
sequencing, although as pointed out by the others
in this thread, desalted oligos are better.
For most applications, you don't actually need
purified oligos. Thus the relative safety in
marketing unpurified (although desalted) oligos
as "purified" since it doesn't make a heck of a
lot of difference.
You should trust your oligo supplier, and your
oligo supplier should respect you. It would be
good for users to challenge their suppliers as to
what is actually meant by ambiguous terms that
have a "marketing" sound to them. God forbid that
scientific research reduces to the level of most
everything else in our society, where anything
may, and possibly does, mean anything. The
conduct of science requires precise terminology.
Demand it, and you will find that you will get it.
(Did any of that help?? I certainly feel better
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