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Hi!
I am trying to figure out the different ways there are to flank completely unknown fragments of DNA (with blunt ends) with adapters, in order to know what primers to use.
I have seen that you can buy adapters (TruSeq) for Illumina sequencing, and think I understand the way these are attach with a T-overhang, and with only 12 nucleotides being complementary between the universal adapter and the index adapter. These are very expensive however, and as far as I understand, they can only be bought as a complete kit for library preparation.
Another company have suggested that I should by Ready-made primers, one forward and one reverse, and than order the compliment sequences to these and use these duplexes as adapters, and then use the same primers as primers. I don't understand how this would work because the way I see it, these duplexes could flank the DNA in different directions, and it the direction is "wrong", the primer would bind to the adapters with its 5' end towards the DNA of interest, and thus the polymerase can't synthesize the fragment because it only synthesizes in the 5'-->3' direction. And furthermore, in many cases, the two primers would bind to the same strand of DNA, leaving the other strand unsynthesized.
Am I wrong about this, or is there a way to direct the adapters to be ligated in the correct direction? Is that why TruSeq adapters have a T-overhang for instance?
Could anyone help me out with this? How does this whole adapter-ligation work, what do you need for it and if there is commercial adapters to buy (except from the TruSeq), where can you buy them from?
I'm knew on this and don't have anyone around me to as, so any suggestions, comments or explanations would be of great help! Thanks!
I am trying to figure out the different ways there are to flank completely unknown fragments of DNA (with blunt ends) with adapters, in order to know what primers to use.
I have seen that you can buy adapters (TruSeq) for Illumina sequencing, and think I understand the way these are attach with a T-overhang, and with only 12 nucleotides being complementary between the universal adapter and the index adapter. These are very expensive however, and as far as I understand, they can only be bought as a complete kit for library preparation.
Another company have suggested that I should by Ready-made primers, one forward and one reverse, and than order the compliment sequences to these and use these duplexes as adapters, and then use the same primers as primers. I don't understand how this would work because the way I see it, these duplexes could flank the DNA in different directions, and it the direction is "wrong", the primer would bind to the adapters with its 5' end towards the DNA of interest, and thus the polymerase can't synthesize the fragment because it only synthesizes in the 5'-->3' direction. And furthermore, in many cases, the two primers would bind to the same strand of DNA, leaving the other strand unsynthesized.
Am I wrong about this, or is there a way to direct the adapters to be ligated in the correct direction? Is that why TruSeq adapters have a T-overhang for instance?
Could anyone help me out with this? How does this whole adapter-ligation work, what do you need for it and if there is commercial adapters to buy (except from the TruSeq), where can you buy them from?
I'm knew on this and don't have anyone around me to as, so any suggestions, comments or explanations would be of great help! Thanks!