TTCCTTGTGTTGGTT
Now my only suggestion to use it as one of the primers on a synthetic DNA.
Oligonucleotide is dissolved in blood samples.
you could try designing primers ending ACGGAAG AND AACCAA with artificial 5'ends such as catx6 and catx 10 and run a very low annealing temperature pcr to generate a product hopefully of 2x primer length plus the missing 2 bases. Hot start pcr would be essential as would controls to make sure primer dimer is very low/non existent. Cutting the product with bsl1 would then give 2 different sizes of product possible separable on PAGE (primer lengths could be manipulated to make visualisation easier. .It depends on how much olriginal oligo there is in your original material compared with existing genomic sequences where the primers sit very close together (probably many). Shame your oligo does not have a biotin tag to make it easy to pull out prior to pcr.
Possibly also make a biotin labelled long oligo (cat x10 ) ending complementary to your sequence and incubate with your sample to bind all of your oligo .pull the bound oligo out with avidin beads. Wash and complete the double stranding of the ss/ds product on the beads. and design primers to the now purified product and its extended oligo and pcr. This might work if your oligo is circulating or in cell culture medium and nterfering sequences on genomic dna do not bind too much of your biotin oligo Hopefully someone comes up with some idea less fraught with non specificity issues than this.good luck
Thanks Paul. I'll think about your ideas. Especially about first one. Problem that method have to be quantitative (I'm sorry, that didn't write it at description).
Original olig will be very much in original material. Before this experiment I worked with a bit longer and more diverse sequence olig, I detected it with some kind of stem-loop PCR and have to dilute material 1000 times to not overload reaction. New olig will be at same amount.
Andrei, thanks, but I think it will be semi-quantive method (or not?). And I can't modify oligo.
ok I still dont know the experimental conditions but if you are diluting greatly to get in range is there any scope for a non pcr method such as binding an antisense oligo Fam labelled and extracting the double stranded product and reading the fluorescence against a standard curve of known amounts of oligo bound to a/s oligo in vitro ( removing the ss antisense non bound oligo with dnase perhaps or maybe not removing the unbound oligo and just reading the coloured peak sizes on a sequecer. again an inert tail would be needed on the binding oligo to bring the size of the product onto the size standard range of the sequencer
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