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Good morning,

I am new to molecular biology. The question might be silly but i would like to know the answer. I have three 60mer single strand synthetic oligonucleotide. Namely Tag 1 - 3. My goal is to assemble these three together by pcr. So i designed two primers having overlap of 18nts from each tag 1 and tag 2 and then Tag 2 and tag 3 ( i mentioned the orientation of my primers). What is the best way to assemble. I read about PCR- based two-step DNA synthesis. But i would like to know more about this.

If you have any questions please feel free to post. Thanks and looking forward for your valuable answers

[the construct i need to assemble]

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In order to assemble your TAG oligos by PCR you will need to redesign your primers so they are complementary to the TAG oligos as DNA polymerases work by adding nucleotides to the 3' end of a DNA strand.

Your design would look like this :

enter image description here

Now with this design you won't be able to assemble everything together as TAG-3 complementarity to primer 2 only presents 5' ends to be extended, which your DNA polymerase can't do. So you can either order the entire reverse complement of your TAG-3 oligo or design a primer 3 that would anneal at the 3' end of TAG-3 to amplify TAG-3.

With all these parts, you should be able to assemble everything in a single reaction, but depending on your downstream process you may have to purify the product of interest as the single reaction will generate intermediates (1, 2, 3, 1+2, 2+3).

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  • $\begingroup$ Really thanks for your explanation. So i have to redesign the primers that should share complementary with tag-1 and tag-2 (not reverse complement right). I will assemble first tag-1 and tag-2 and then i will go for tag-3 with my product. $\endgroup$ – Rengaraj Mar 2 at 19:17
  • $\begingroup$ You should order the reverse complement of your primer 1 and 2. Let's say that your TAG-1 sequence is 5'-attgcctg-3' and TAG-2 sequence is 5'-TAATTGCC-3'. So far what you have ordered as primer 1 is 5'-ctgTAA-3' but you need the reverse com 5'-TTAcag-3' $\endgroup$ – GaelC Mar 2 at 19:33
  • $\begingroup$ Awesome. Once again thanks. $\endgroup$ – Rengaraj Mar 2 at 19:43
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    $\begingroup$ @Rengaraj If this answer addressed your problem, please consider accepting it by clicking on the check mark/tick to the left of the answer, turning it green. This marks the question as resolved to your satisfaction, and awards reputation both to you and the person who answered. If you have >= 15 reputation points, you may also upvote the answer if you wish. There is no obligation to do either. $\endgroup$ – MattDMo Mar 3 at 20:51
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I would suggest using Gibson assembly. I am not an expert but this is the kind of problem that it is designed to solve. Probably there is some way to do this by PCR but I expect that it will be quite finicky.

See this quote from that page (my emphasis):

Tip: “Stitching” Fragments Together using Oligos

When you need intervening sequence between two PCR products, one method is to “stitch” together several oligos. This technique is especially useful for introducing promoters, terminators, and other short sequences into the assembly and is used when the part to be inserted is too long to include on overlapping PCR primers (>60 bp) but too short to make its own part (<150 bp).

Please note that the way to design the “stitching” primers and the amounts of them to include in the Gibson reaction are different than with normal PCR primers. The details are published in ( Nat Methods 2010; 7:901-3 ).

Tip: Number of Fragments Assembled Simultaneously

Multiple fragments can be assembled in one reaction. However, some labs have observed a sharp decrease in success rate when assembling more than 5 fragments at a time.

Update

It looks like there are people who argue both that Gibson is impossible with short fragments or that it works fine. There is more information on this here and here and here. Due to the relatively complex nature of your reaction, I think that Gibson might save you time. But other people linked there do claim that PCR works better.

Update 2

GaelC's answer above has better/more specific methodological advice on PCR assembly.

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  • $\begingroup$ I agree with Gibson assembly but i read that it is not suitable for short fragments such as 60-mers. $\endgroup$ – Rengaraj Mar 2 at 18:24
  • $\begingroup$ @Rengaraj see update $\endgroup$ – Maximilian Press Mar 2 at 18:44
  • $\begingroup$ Exactly, these are the posts i went through. First i will check by PCR assembly, if it did not work i will order the Gibson assembly kit. And i have a doubt? the way i designed the primers are fine or correct? or should i have to change anything... $\endgroup$ – Rengaraj Mar 2 at 19:02
  • $\begingroup$ @Rengaraj I think that GaelC's answer is good if you choose to go with PCR. $\endgroup$ – Maximilian Press Mar 2 at 19:07
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    $\begingroup$ NEB's tech support is fantastic. If anyone has the answer about Gibson assembly of shorter sequences, they would. $\endgroup$ – MattDMo Mar 3 at 21:01

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