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Is there a way to extend a small fragment of DNA, say 150 bp, by making copies of itself and attaching each copy of that small fragment to the end of that 150 bp sequence?

For example, I want a 1 kbp+ DNA fragment made up of copies of that exact 150 bp sequence.

I am performing optical tests on DNA strands, but the issue is that the DNA fragments that I am targeting are too small.

However, if the DNA fragment is just a repeated sequence of the DNA fragment I am testing, the tests will yield the same results. For that reason, I am trying to create a long strand of DNA from just one DNA fragment copied over and over.

Is there a procedure that I can follow to accomplish this?

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  • $\begingroup$ well you can use PCR in conjunction with some ligation technique... $\endgroup$
    – TanMath
    Commented Aug 30, 2015 at 20:12
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    $\begingroup$ What exactly are you trying to do? If you give a clearer explanation of your experimental setup and goals, we might be able to help you further. As it is, this question is rather vague. $\endgroup$
    – MattDMo
    Commented Aug 31, 2015 at 1:02
  • $\begingroup$ @MattDMo Hi, basically I am performing optical tests on DNA strands but the issue is that the DNA fragments that I am targeting are too small. However, if the DNA fragment is just a repeated sequence of the DNA fragment I am testing, the tests will yield the same results. For that reason, That I am trying to create a long strand of DNA from just one DNA fragment copied over and over. $\endgroup$ Commented Sep 3, 2015 at 0:00
  • $\begingroup$ If the both ends satisfy the condition under which ligase works, you just treat with ligase. But you get various length of ligation products. You can cut from agarose after electrophoresis if you think the yield is enough. $\endgroup$
    – 243
    Commented Oct 2, 2015 at 0:45
  • $\begingroup$ Could you provide some more details? Does your fragment have blunt ends? Is the entire fragment important or could you cut in a little bit if need be? $\endgroup$
    – AMR
    Commented Oct 2, 2015 at 1:53

3 Answers 3

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If what you trying to get is 1kbp of repetitive 150bp sequences, then synthesis as a large chunk might be better idea. It costs ~150-300$ but will potentially save tons of time. (see gBlock from IDT in US)

PCR and ligation with repetitive sequences is pain the butt. It might be true that you save money, but time and frustration could be more expensive.

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  • $\begingroup$ IDT doesn't like highly repetitive sequences, they become hard to do quality control on. It depends on your exact sequence though, so put your sequence in their form and see if it comes back with a problem. If not, this is probably a good cheap way to do it. Be sure to get PCR primers for your sequence too, so you can make more. $\endgroup$
    – user137
    Commented Jun 30, 2016 at 3:20
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Given the cost or DNA synthesis you could probably order the two single strands and anneal them together. Engineer two complementary restriction enzyme overhanging ends onto the sequence. For example, at one end engineer a partial Bam HI site and on the other end engineer a Bgl II site. Those sticky ends will anneal but the resulting ligation product cannot be cut by either enzyme.

By alternating cycles of ligation, digestion, and ligation, you can build up concatemers of your starting fragment. Then you can gel purify the longer pieces and sub-clone them into a suitably prepared vector.

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  • $\begingroup$ Instead put adaptors? (if orientation is not an issue) $\endgroup$
    – Dexter
    Commented Dec 1, 2015 at 17:56
  • $\begingroup$ If orientation is not an issue you can use a blunting enzyme or a blunt-end-polymerase during the PCR (like Phusion polymerase) and then go for a blunt-end ligation. N.B. primers need to be phosphorylated! $\endgroup$
    – alec_djinn
    Commented May 30, 2016 at 14:25
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I suggest you to try Recursive Directional Ligation. It is meant to do exactly what you are aiming for, i.e. to "polymerize" short DNAs into a longer one. You can find the protocol here http://pubs.acs.org/doi/abs/10.1021/bm015630n

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