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    • #6587
      SororSaudade
      Participant

      Hello

      Does anyone know if it is posible to do a PCR amplification of a small fragment with only one primer (that anneals only at one site)?
      Plus, ideas to separate two possible types of PCR products are more than welcome 8)

      Thanks a lot!

    • #63236
      Oscerot
      Participant

      Well, you see, PCR is used to make an exponential amount of copies of a specific gene. Since genes usually have a complimentary bases in order to work, it would seem impossible to successfully do PCR with one primer on one strand of the DNA.

      A way to seperate two types of products is the gel electrophoresis method (or it may be called "gel electrolysis").

      ====

      On a side note, it may be very beneficial to you to know that my knowledge on the subject so far only extends to Grade 12 Academic Biology. ๐Ÿ˜†

    • #63240
      SororSaudade
      Participant

      eheh I know what PCR and electrophoresis are!
      Just asked because i could be missing some points and nowadays there are several types of PCR for different purposes.

      about electrophoresis… I forgot to mention that I want to separate two products with exactly the same size… ๐Ÿ˜›

      thanks ๐Ÿ™‚

    • #63245
      Oscerot
      Participant

      Oh, haha.

      Use your fingers silly!

    • #63268
      SororSaudade
      Participant

      how nice of you… ๐Ÿ˜†

    • #63279
      Shallos
      Participant

      i think it is impossible to amplify a gene with only one primer…
      or maybe i don’t get your meaning…

    • #63303
      canalon
      Participant

      You can amplify a gene with one primer, if you prime with a sequence located in invrted repeats urrounding your fragment of interest; It is not that frequent though. And in all other case you are going to produce little DNA , and of plenty of different sizes, so it is not going to be very useful.

      As for separating fragments of identical sizes, but different sequences, it can be done using gradient gel electrophoresis (DGGE or TGGE) protocols can easily be googled.

    • #63343
      SororSaudade
      Participant

      that’s what I thought… PCR with one primer that anneals at only one site is generally used for sequencing, but not to amplify fragments.

      I really didn’t know those types of electrophoresis.

      Thank you very much ๐Ÿ™‚

    • #63407
      wbla3335
      Participant

      Depends on what is a "small fragment". If it’s a small fragment of a chromosome, it could still be quite large. If it’s a PCR product or restriction fragment of reasonable size, you can do a PCR-like reaction with one primer to linearly (not exponentially) amplify one strand of the fragment. This is commonly done to, for example, incorporate a label into the amplified strand for detection (as in one method of SSCP).

    • #63417
      SororSaudade
      Participant

      thank you all for your comments, the were very helpful ๐Ÿ™‚

    • #68703
      hara
      Participant

      i think that if you want to seperate two pcr products with exactly the same size the next step after electrophoresis is to do a digestion of the pcr products with a restriction enzyme that cut the two sequences to different sites (so different fragments) and then do electrophoresis of the uncuts and the cuts…so you will know what each product is …(this of course you know it before you do your pcr) but this is a way to persuade someone else what each product is…and of course an other way if the size is not exactly the same but somes bp different is to use not an agarose gel but a polyacrylamide gel….

    • #68718
      SororSaudade
      Participant

      unfortunately the size is exactly the same and the sequence only differs in about 10 nucleotides (in a 1,8kb fragment) and there won’t be new restriction sites.
      well… I think I just found another way to do what I want, by linearly amplify one strand and purify it… then, after the PCR, I will only get one type of product (I haven’t tried it yet, though :P)

      Thanks for your help anyway!

    • #69517
      muraceae
      Participant

      Forรงa aรญ pekena!!! tu vais conseguir!!! Inyfffeas!!!!!!!!!!

    • #69518
      SororSaudade
      Participant

      olha… tinรณnis pra ti!!!!!!!!! ๐Ÿ˜†

      nha nha nha nha nha

    • #103938
      Campylobacter
      Participant

      well here is a thought…

      my thrid year project was based on desiging primers to identify a specific gene of a genomic DNA but i didnt order the compliment of the reverse primer, therefore i was left with two forward primers ( if that makes sense :P)… however the funny thing was i was able to get three different PCR products.. one of the pcr prodcuts i was able to rule out because it was bigger that target gene… however the other two pcr products could be a possibility…. i got one of the two pcr products sequenced and it turns out to be the target gene i was aiming for and this pcr product was about 400bp in lenght… after bioinformatic analysis… the only possible conclusion was that my forward primer is acting both as the reverse and the forward!!! all 23bases of the forward primer binds at one end ( so that is the forward primer) however only 7 bases bind at the down stream ( reverse primer)… the Tm of the 7bases turned out to be 32C and the annealing temp was at 55 C?? IS it possible… but interesting though.

      well i suppose in away its possible… but to get very clear results i.e to get A band as oppose to multiple bands… you do need two primers forward and reverse because otherwise u will get lot of products of single stranded DNA which is not enough to be detected by gels… therefore double stranded PCR products are required.

    • #103939
      JackBean
      Participant

      the problem is not with ss/dsDNA, but with the quantity, which is increasing linearly in case of ssDNA and quadratically in case of dsDNA.

      I could believe, you got your product, if your primer’s most-3′ 7-in-a-row nucleotides annealed to the reversed strand. But you should better check that before.

    • #103943
      canalon
      Participant

      Well, the first few cycles will give very poor results indeed, but the thing is that once you start having enough amplicon from the template, those amplicon will fully match your primers, because they have included the primer at that time, and they will amplify exponentially.

      I guess you have been very lucky that this 7 nt motif was specific enough, because amplification with non speicific primers, although possible, is not something very reliable. even more so when it comes to the final product. I guess a high number of cycles, a very specific forward primer, and a 7 nt motif rare enough not to ever anneal at a distance close enough to generate non specific product within your Paramaters all conspired in your favor. You are very lucky.

    • #104042
      JackBean
      Participant

      yeah, canalon is definitely rigth, I didn’t think of that. Once you get some mis-amplicon, it will grow exponentially anyway

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