I want to make double stranded DNA out of single strands using random decamers or other lengths of random oligos. The conditions is that the random oligos should space apart about 1 kb to 1.5 kb apart. I need suggestions on the sequence so that the coverage is maximized because the sequence of single stranded template is not known.
You could use random hexamers to get the double strand DNA and then digest with a suitable restriction enzyme, or sonicate to shear the DNA, to 1-1.5 kbp
Good luck.
Hi Steven,
Do you think that the random hexamers would extend to >2kb? If yes, then using which polymerase? Also, I need the ends to be blunt so a complete extension is required. Can you suggest more?
The average binding of a random hexamer would be once every 46 bases i.e 4.1kb. Because the oligos are random, the location and size between oligo binding sites will be variable with an avg of between 1024-4096 bases (5 and 6 mer). There is no method of getting exact sizing.
Hi Ian,
I think in a random hexamer where each base can be any 1 out of 4, it will be 16 = 1. According to this, hypothetically each random hexamer can land next to each other, or even overlap with each other. Your calculation will apply if each base is defined in which case it is only 1 out of 4. However all of this is an estimation and the final annealing will depend upon target sequence complexity, bias in the synthesis of random sequence etc. I can only estimate by designing a mixed base, degenerate oligo, but as you said, it will never be exact sizing.
My correction. I misread/ misunderstood the original point. I was thinking not of random hexamers , but of sequence specific primers / enzyme recognition sites. It should be possible to design a hexamer that contains a region of defined sequence (eg 4 bases) and one of degeneracy (eg 2 bases). Using RE sites as an example, the probability that a random 6-mer matches the EcoRI binding site is (1/4)6, so the site occurs about once every 46 (= 4,096) bp in a random DNA sequence. The HindII binding site, on the other hand contains two positions where two out of four bases can match (GTYRAC), would occur once per 44 x 22 (= 1,024) bp. Using this hybrid hexamer one should in theory prime once every kb.
....on the other hand I could be talking B/S.......
Hi Ian,
Thanks for the tip. I have already designed and ordered semi-specific or semi-degenerate random decamers. I will be using them on human genomic DNA, and now the problem is how much will be the coverage. However I will know this only after a pilot experiment.
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