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I've found this paper which involves the construction of 19-bp random DNA sequences, but I don't know enough biology to understand how this method works. Could someone explain it to someone who is highly technical, but has knowledge of only introductory, undergraduate biology? How do the authors know that their resultant sequence is truly random?

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If you want to synthesize a specific DNA sequence chemically, you start by attaching your first nucleotide (one letter in your DNA) to a solid phase. You can then continue adding individual letters to it. Because it is bound to a solid support material, you can wash the whole thing without washing your growing DNA away.

So if you want to synthesize AGC, you would first only add A to your reaction and then wash away all the A that hasn't reacted. Then you'd add G, which binds to your previous A, and again wash away all the G that hasn't reacted. Then you'd add C, and do the whole thing again.

If you want to create a random or semi-random sequence, you simply add multiple letters at once. If you always add all four letters in each step, you'll get a completely random sequence. You can also randomize only specific positions, and only there add all letters.

The result is random, but I don't think it is as random as e.g. a cryptographically secure pseudorandom number generator. There are various factors that can affect the randomness, variations in the concentrations of your nucleotides, variations in the quality or composition of your reactants and probably a lot more.

See also Oligonucleotide synthesis on Wikipedia

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  • $\begingroup$ They say that the maximum sequences is 4¹⁹ which means every permutation. So I am not sure if they really synthesized random bases per each position or just synthesized all possible variants by allowing equimolar mixture of nucleotides at every step. This is what you also describe but the result IMO cannot be called random. It is expected to yield all permutations. $\endgroup$
    – WYSIWYG
    Commented Dec 14, 2015 at 8:50
  • $\begingroup$ @WYSIWYG I'll have to look at the paper again, I though I had read them indicating that they were using chemical synthesis like I described, but it looks like I read that into their description $\endgroup$
    – Mad Scientist
    Commented Dec 14, 2015 at 8:53
  • $\begingroup$ This is what is written in the M&M: "Random sequences were synthesized with equimolar mixtures of phosphoramidites." This is exactly what you said but I am not sure if that can be called random, in the sense of RNG. $\endgroup$
    – WYSIWYG
    Commented Dec 14, 2015 at 8:56
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    $\begingroup$ @WYSIWYG Each individual coupling is random, and if you get to a length of DNA where you don't have enough molecules for all permutations, it gets truly random as well. $\endgroup$
    – Mad Scientist
    Commented Dec 14, 2015 at 9:01
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    $\begingroup$ For practical purposes, IDT offers randomized oligos. Saves a lot of trouble.Other companies probably offer this too. $\endgroup$
    – user137
    Commented Dec 14, 2015 at 14:47

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