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Introduction of restriction sites via extended primers?

Introduction of restriction sites via extended primers? - Protocols and Methods Forum

Introduction of restriction sites via extended primers? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-04-2004, 02:04 PM
Peter Frank
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Default Introduction of restriction sites via extended primers?



Hello,

I want to introduce new restriction sites to the ends of a DNA
sequence via extended primers, i.e. the primers will have a matching
region (around 20 bases) and then the non-complementary restriction
site (around 6-8 bases).
I found a source saying "Extra bases are usually added to the 5' end,
as restriction enzymes do not cleave DNA efficiently at the end of a
fragment.".

How many extra bases are usually added and does it matter which ones?
Are there any other criteria that should be considered when designing
primers for introducing new restriction sites by PCR?

Peter
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  #2  
Old 08-04-2004, 06:11 PM
Michael L. Sullivan
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Default Introduction of restriction sites via extended primers?

Oligos are cheap, so I usually add 6 extra bases of "GC-clamp", that
is GCGCGC. Presumably G and C are preferred because of the higher
melting temperature they'd impart to the ends. This strategy has
generally worked well for me. Of course, if you were introducing
GCGCGC as your restriction site, something else might be better of
the bases on the end.

One thing to keep in mind if you add any extra bases on the other
side of the restriction site (if for example you are adding a
translational terminator or other desired sequence), make sure you
don't introduce a methylation site. It won't matter in the PCR
product, but once the fragment is cloned, it'll be trouble!

Mike



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  #3  
Old 08-13-2004, 02:10 PM
salli
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Default Introduction of restriction sites via extended primers?


"Kyle Legate" <[Only registered users see links. ]> skrev i en meddelelse
news:[Only registered users see links. ]...
from
an
certain


Hi Kyle.
I don't understand the last bit, where you say that AATT contributes less to
annealing that G and C? This is of cause correct, however this overhang is
NOT going to anneal to template - so do you include it when calculating
annealing temp?
Just wondered
Salanti.


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  #4  
Old 08-13-2004, 06:06 PM
Peter Ellis
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Default Introduction of restriction sites via extended primers?

[Only registered users see links. ] wrote:


It doesn't anneal to template in the first cycle, but it will do for
all subsequent cycles - you want the difference to be as little as
possible, hence using A's and T's.

Peter
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  #5  
Old 08-13-2004, 06:07 PM
Peter Frank
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Default Introduction of restriction sites via extended primers?

"salli" <[Only registered users see links. ]> wrote:


I am not the one who gave the answer but the one who started the
thread but I think what Kyle meant was that AATT contributes less to
the melting temperature of the original primers annealing to each
other. For the primer-template hybrids the overhang will be irrelevant
and not affect the annealing temperature. However, pimer dimers may be
more likely with GC-rich overhangs than with AT-rich overhangs because
as you know GC base pairs cause stronger binding than AT base pairs.

Peter
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  #6  
Old 08-13-2004, 09:54 PM
Peter Frank
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Default Introduction of restriction sites via extended primers?

I wrote:


I have to correct myself or at least be more specific. It will not be
relevant for the very first primer-template hybrids with the template
being the molecules one introduced into the PCR mix.
But for newly synthesized amplicons serving as template it will affect
the annealing temperature.

Peter
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  #7  
Old 08-14-2004, 06:34 AM
salli
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Default Introduction of restriction sites via extended primers?

OK, thanks for clarifying. I do understand it now :-)
"Peter Frank" <[Only registered users see links. ]> skrev i en meddelelse
news:[Only registered users see links. ]...


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