IPCR

Doug Rhoads DRHOADS at MERCURY.UARK.EDU
Sun Jul 31 10:33:16 EST 1994


>To get junction fragments beyond a region of known sequence, universal PC=
R
>can be used as an alternative to inverse PCR.  We have used this approach
>to clone  insertion junctions of a retrotransposon.  The method is based 
>on one described by Sarkar et al. in PCR Methods & Applications 2:318-322
>(1993), and worked beautifully for us on the first try.  Briefly, you nee=
d
>to make two nested "forward" primers based on a region near the end of 
>your
>known sequence, with the 3' ends pointing out toward the unknown flanking
>region.  The universal "reverse" primer has the following design:
>5'-T7-X-NNNNNNNNNNGATC-3', where T7 is the phage promoter sequence, or an=
y
>other specific sequence not present in the template DNA, X is an optional
>restriction endonuclease recognition sequence to facilitate subsequent
>cloning of the amplified fragments (we omit this, since we do TA cloning)=
,
>the next 10 nt's are completely degenerate, and GATC is any specific 4-6 
>nt sequence (the restriction recognition sequence could be put here rathe=
r
>than next to T7).  Do two rounds of nested PCR, the first using forward
>specific primer 1 and the universal reverse primer.  Then take a 1 |l
>aliquot as the template for the second round of PCR, this time using
>forward primer 2 and T7 as reverse primer.  Gel-purify the individual 
>bands (you will probably get several bands from each reaction), clone and
>sequence, using forward specific primer 2 as sequencing primer.  Good 
>luck. -- 
>Dick Beeman
>beeman at crunch.usgmrl.ksu.edu
>"One of the advantages of being disorderly is that one is constantly 
>making exciting discoveries".  A. A. Milne
>

This post brings up a question I have often wondered about.  When does one=
 
decide to add extra of a degenerate primer.  The `universal' primer Dr. 
Beeman describes would have an approximate million-fold (4^10=3D1,048,576)=
 
degeneracy.  We have worked on amplifying homologous gene regions based on=
 
degenerate primers developed from reverse- translation of conserved 
protein domains.  Usually when we have 30 or 40,000 fold degeneracies we 
up the primer 2-3x (from around 0.8 uM in the reaction).  In other tests 
with specific, non-degenerate primers we have obtained much more 
specificity in PCRs when we drop the primer concentrations to around 0.1 
uM.  But with degenerate primers such as the `universal' primer, after the=
 
first cycle, don't you generate a million-fold excess of potential 
competitive inhibitors for the priming site, plus reduce the concentration=
 
of effective primers a few thousand fold??  Granted that by allowing 
single base differences you can mutate the amplicons at every cycle until 
virtually `every' primer can be utilized, but that doesn't help in the 
early cycles.  If one assumes single base mismatches (I know this is 
errored since the position of the mismatch relative to the primer 3' end 
will be important) are allowed in the second annealing cycle then 
isn't the amount of effective primer severely reduced.  I may get 
deservedly flamed by the statisticians but isn't it about 262,000 (4^9) 
of the 1,000,000 (4^10) possible primers??  Sorry, its Sunday AM and that 
statistical leap may be wrong.  
Any one out there with an opinion on this one??



Doug Rhoads                  || Dept. of Biological Sciences
drhoads at mercury.uark.edu     || 601 Science Engineering
drhoads at uafsysb.uark.edu     || University of Arkansas
501-575-3251                 || Fayetteville, AR 72701



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