PCR

Andy Wang andywang at VAXA.WEEG.UIOWA.EDU
Thu Nov 11 09:31:29 EST 1993


Dear Patrick,

   This is a belated thank you note for sending me Gulliver probes. I
apologize for not acknowledging it sooner. I have not had a chance to try
it yet, because insertion mutagenesis in my lab started to work well just
at the time the probes arrived and I have been busy sorting the mutants
out. Thanks again and good luck in your search for homologs of mating
related genes.

Andy WangDear fellow Chlamy netters, A month or so ago, I sent out an open request for 
information on using degernate primers to do PCR with Chlamy RNA. I received 
quite a few responses. I think I have acknowledged all responders. However, 
because I don't know how to keep record of out-going mail, I have no way of 
checking and would like to apologize for any misses. I put all the answers 
together with a word processor and am now send it out for general 
distribution. I hope the information will be of some use to some people. 

Andy Wang
andywang at vaxa.weeg.uiowa.edu

 We used PCR with degenerate primers to amplify a piece of a Phospholipase C 
in Chlamydomonas eugametos. We had three primers, one towards the 3' and two 
towar ds the 5'. So they could be used in two reactions. Because the fragments 
had to  overlap, the larger fragment should recognize the shorter one in a 
hybridizati on study. The reactions gave a lot of bands (just as you 
describe), even in the case when  used in wrong combinations or if we used 
just one. In the reactions which shou ld give the desired product, we saw a 
band of the expected size. In a hybridiza tion study the larger one indeed 
recognized the shorter one and therfor the lar ger one was cloned and 
sequenced. Sequencing revealed that in the isolated PCR- band from agarose 
gel, there were at least three different sequences with no ho mology to each 
other except the primer sites. One of the sequences had a very high homology 
with the animal PLCs so we succeeded! We believe that it is important to use a 
"nested" primer approach (so more than two primers which have to give products 
which recognize each other) and that you have to sequence more than just one 
clone with the PCR-product. Hope this helps, if you have further questions 
please ask. Henk van de Kamer CHLAMY at SARA.NL      


  We used once rePCR and ended up the same as you. Lot of not wanted 
sequences. After improving the PCR (DNA concentration and primer concentration 
proved to b e essential and must be optimized for each primer) we got the 
wanted band in th e first round of PCRing. It still had a lot of not wanted 
sequences, but we got  also the right one. Sometimes we believe that you can 
better use 25 cycles than the standard 30, pr obaly because the last 5 cycles 
the enzym is more prone to errors. We don't hav e exclusive prove of it, but 
may be it is worth to try. The annealing temperature is dependant on the 
calculated melting temperature of  the lowest primer. Usaly we take 2-5 
degrees lower as the calculated one. The concentration of the primers is in 
the order of 100 pM, but is dependant on the  degenaracy. Hope this helps or 
else lett me know, Henk   


My laboratory has usedd degenerate oligos to PCR a serine protease from 
Chlamy. The oligos were designed from the consensus sequences around two of 
the three amino acids of the catalytic triad of serine proteases.  WE got the 
primers from Dave Banfield (then at Dept. Biochem. Univ. Brit. Columbia, 
Vancouver, Canada T-604-228-4698), who designed them almost identically to 
Sakanari et al as described in PNAS 86: 4863, 1989. We got one strong band 
using genomic DNA and when we cloned and sequenced the product we found the 
third, internal consensus sequence expected for serine proteases. We were 
unsuccessful starting with RNA.  I believe that Pete Lefebvre of U. Minn. and 
Joel Rosenbaum' s lab at Yale have used PCR with Chlamy.  Please call, write 
or e-mail us if you want mor e information.  
T-214-688-2349;FAX-214-688-8694:e-mail-Snell03 at swmed.  Yours, Bill  Snell 
UT-Southwestern Med. Ctr., Dept. Cell Biology and Neuroscience, 5323 Harry 
Hines  Blvd. Dallas, TX, 75235-9039


In reply to your request for information on using degenerate primers with 
Chlamy DNA:  yes, I've done it successfully.  The primers were based on highly 
conserved sequences between three green algae (including Chlamy) and used to 
isolate a sequence from a fourth alga.  Chlamy was used as a control for the 
PCR reaction.  The primers were degenerate, but not very (one had a degeneracy 
of 4 and the other had a degeneracy of 16), so this may not be very helpful to 
you. Anyway, I used fairly standard conditions, but it was important to add 
20% glycerol to the reaction mix; annealing temp was 45 C (although I had some 
luck at 55 C).  Good luck. 

 Terri Dunahay National Renewable Energy Lab Golden, Colorado     


 Yes, I have successfully amplified Chlamydomonad genes using degenerate 
primers.  For my PCR conditions, I found that the most critical parameter for 
reducing the number of spurious bands was the Mg++ concentration. Small 
changes (1.0 mM --> 1.5 mM --> 2.0 mM) can make the difference between no 
bands, one band, or greater than five bands in some reactions...  Before 
varying any of the reaction conditions, though, I'd recommend doing a quick 
southern on your PCR products, probing with a gene analogous to the one you're 
hoping to amplify.  If you have access to such a probe, this will let you know 
which of those many bands is the one you're after.  I hope that's helpfull.  
If you have any questions, feel free to contact me by email or phone!  
-Peter Kuhlman pkuhlman at bio.indiana.edu (812) 855-2549  
Palmer Laboratory 
Indiana University  


We find that decreasing the Mg ion concentrations eliminates some bands. 
However, especially with very degenerate primers, multiple bands are sometimes 
unavoidable.  Our lab is experiencing difficulties with multiple bands as 
well.  Perhaps if you could give us the specifics of your protocol we may be 
able to give some suggestions.  We are interested in what you have tried as it 
may aid us in our search.  Please don't hesitate to contact us on details, or 
information on other PCR books that we use in our lab.  I am Scott Hoffman, a 
graduate student and can be reached at Spanier at Badlands.NoDak.Edu  
21-SEP-1993 07:53:53.99                       


Hi,
I do not know,  h o w  degenerate your primers are, but we used several
degenerated primer pairs designed to find common domains of G proteins,
and they worked nearly all quite nice. However, I should mention that the
template was not a genomic DNA but cDNA (1st strand synthesized
from total RNA). If this info is of any use for you, and you need some more 
details,
please contact me again.

Stefan Fabry
(University of Regensburg, Germany)

concerning your further questions I can say, that most of our primers are 
degenetated to the same extent, some even more. We got in most cases only one 
prominent band, but this was also somewhat dependent on the stringency of 
annealing. We do not clone a special band cut from the gel, but just clone the 
whole PCR reaction (we developed a novel, very efficient method doing that, 
that appeared in Nucl. Acid Res. at beginning of August this year). We then 
select on the level of individual clones (rapid plasmid analysis). Most clones 
have appropriate inserts. When sequencing individual clones, we always get 
several different sequences. They normally fall into the main classes of G
proteins, as expected, however, we always find also some completely unrelated 
sequences, too. These were discarded.  If additional information is necessary,  please feel free to contiunue our dialog!  Stefan Fabry Dear fellow Chlamy netters, A month or so ago, I sent out an open request for
information on using degernate primers to do PCR with Chlamy RNA. I received 
quite a few responses. I think I have acknowledged all responders. However, 
because I don't know how to keep record of out-going mail, I have no way of 
checking and would like to apologize for any misses. I put all the answers 
together with a word processor and am now send it out for general 
distribution. I hope the information will be of some use to some people. 

Andy Wang
andywang at vaxa.weeg.uiowa.edu

 We used PCR with degenerate primers to amplify a piece of a Phospholipase C 
in Chlamydomonas eugametos. We had three primers, one towards the 3' and two 
towar ds the 5'. So they could be used in two reactions. Because the fragments 
had to  overlap, the larger fragment should recognize the shorter one in a 
hybridizati on study. The reactions gave a lot of bands (just as you 
describe), even in the case when  used in wrong combinations or if we used 
just one. In the reactions which shou ld give the desired product, we saw a 
band of the expected size. In a hybridiza tion study the larger one indeed 
recognized the shorter one and therfor the lar ger one was cloned and 
sequenced. Sequencing revealed that in the isolated PCR- band from agarose 
gel, there were at least three different sequences with no ho mology to each 
other except the primer sites. One of the sequences had a very high homology 
with the animal PLCs so we succeeded! We believe that it is important to use a 
"nested" primer approach (so more than two primers which have to give products 
which recognize each other) and that you have to sequence more than just one 
clone with the PCR-product. Hope this helps, if you have further questions 
please ask. Henk van de Kamer CHLAMY at SARA.NL      


  We used once rePCR and ended up the same as you. Lot of not wanted 
sequences. After improving the PCR (DNA concentration and primer concentration 
proved to b e essential and must be optimized for each primer) we got the 
wanted band in th e first round of PCRing. It still had a lot of not wanted 
sequences, but we got  also the right one. Sometimes we believe that you can 
better use 25 cycles than the standard 30, pr obaly because the last 5 cycles 
the enzym is more prone to errors. We don't hav e exclusive prove of it, but 
may be it is worth to try. The annealing temperature is dependant on the 
calculated melting temperature of  the lowest primer. Usaly we take 2-5 
degrees lower as the calculated one. The concentration of the primers is in 
the order of 100 pM, but is dependant on the  degenaracy. Hope this helps or 
else lett me know, Henk   


My laboratory has usedd degenerate oligos to PCR a serine protease from 
Chlamy. The oligos were designed from the consensus sequences around two of 
the three amino acids of the catalytic triad of serine proteases.  WE got the 
primers from Dave Banfield (then at Dept. Biochem. Univ. Brit. Columbia, 
Vancouver, Canada T-604-228-4698), who designed them almost identically to 
Sakanari et al as described in PNAS 86: 4863, 1989. We got one strong band 
using genomic DNA and when we cloned and sequenced the product we found the 
third, internal consensus sequence expected for serine proteases. We were 
unsuccessful starting with RNA.  I believe that Pete Lefebvre of U. Minn. and 
Joel Rosenbaum' s lab at Yale have used PCR with Chlamy.  Please call, write 
or e-mail us if you want mor e information.  
T-214-688-2349;FAX-214-688-8694:e-mail-Snell03 at swmed.  Yours, Bill  Snell 
UT-Southwestern Med. Ctr., Dept. Cell Biology and Neuroscience, 5323 Harry 
Hines  Blvd. Dallas, TX, 75235-9039


In reply to your request for information on using degenerate primers with 
Chlamy DNA:  yes, I've done it successfully.  The primers were based on highly 
conserved sequences between three green algae (including Chlamy) and used to 
isolate a sequence from a fourth alga.  Chlamy was used as a control for the 
PCR reaction.  The primers were degenerate, but not very (one had a degeneracy 
of 4 and the other had a degeneracy of 16), so this may not be very helpful to 
you. Anyway, I used fairly standard conditions, but it was important to add 
20% glycerol to the reaction mix; annealing temp was 45 C (although I had some 
luck at 55 C).  Good luck. 

 Terri Dunahay National Renewable Energy Lab Golden, Colorado     


 Yes, I have successfully amplified Chlamydomonad genes using degenerate 
primers.  For my PCR conditions, I found that the most critical parameter for 
reducing the number of spurious bands was the Mg++ concentration. Small 
changes (1.0 mM --> 1.5 mM --> 2.0 mM) can make the difference between no 
bands, one band, or greater than five bands in some reactions...  Before 
varying any of the reaction conditions, though, I'd recommend doing a quick 
southern on your PCR products, probing with a gene analogous to the one you're 
hoping to amplify.  If you have access to such a probe, this will let you know 
which of those many bands is the one you're after.  I hope that's helpfull.  
If you have any questions, feel free to contact me by email or phone!  
-Peter Kuhlman pkuhlman at bio.indiana.edu (812) 855-2549  
Palmer Laboratory 
Indiana University  


We find that decreasing the Mg ion concentrations eliminates some bands. 
However, especially with very degenerate primers, multiple bands are sometimes 
unavoidable.  Our lab is experiencing difficulties with multiple bands as 
well.  Perhaps if you could give us the specifics of your protocol we may be 
able to give some suggestions.  We are interested in what you have tried as it 
may aid us in our search.  Please don't hesitate to contact us on details, or 
information on other PCR books that we use in our lab.  I am Scott Hoffman, a 
graduate student and can be reached at Spanier at Badlands.NoDak.Edu  
21-SEP-1993 07:53:53.99                       


Hi,
I do not know,  h o w  degenerate your primers are, but we used several
degenerated primer pairs designed to find common domains of G proteins,
and they worked nearly all quite nice. However, I should mention that the
template was not a genomic DNA but cDNA (1st strand synthesized
from total RNA). If this info is of any use for you, and you need some more 
details,
please contact me again.

Stefan Fabry
(University of Regensburg, Germany)

concerning your further questions I can say, that most of our primers are 
degenetated to the same extent, some even more. We got in most cases only one 
prominent band, but this was also somewhat dependent on the stringency of 
annealing. We do not clone a special band cut from the gel, but just clone the 
whole PCR reaction (we developed a novel, very efficient method doing that, 
that appeared in Nucl. Acid Res. at beginning of August this year). We then 
select on the level of individual clones (rapid plasmid analysis). Most clones 
have appropriate inserts. When sequencing individual clones, we always get 
several different sequences. They normally fall into the main classes of G
proteins, as expected, however, we always find also some completely unrelated 
sequences, too. These were discarded.  If additional information is necessary,  please feel free to contiunue our dialog!  Stefan Fabry 




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