fuzzy questions about DNA strand

Question

[mangfu100]

hello

I am just reading a web page related about dna strand to understand dna strand.(I think it is so confused..)

but I can't understand the context of web pagel.

below is the context of web pages i searched in.

(I can't figure out the second paragraph... why reading both of the strand from respective 3' ends at one result the uninterpretable?)

Does anyone help me understand the below contents?

Sequencing by synthesis, which is how most commercially available high-throughput sequencing technologies work as of December 2012 (see notes on sequencing technologies), always synthesizes the new strand (which becomes your read) in a 5'-to-3' direction. That's because this is how DNA polymerase works in our cells (indeed, in every living thing's cells) and sequencing relies on DNA polymerase. Since the new strand is synthesized 5'-to-3', you are working your way up the template strand in a 3'-to-5' direction.

In any sequencing technology, you PCR amplify the individual DNA fragments once they have hybridized to flowcells or beads. This means you end up with both strands of DNA. If you were to read both of the strands from their respective 3' ends at once, you'd be getting two different sequences and your results would be uninterpretable. To avoid this problem, sequencing technologies ligate non-complementary adapters to the 3' and 5' ends of DNA fragments so that the primer for one adapter only begins synthesis on one strand and not on its complement.

Answer 1

[coleman_jonathan]

DNA is double-stranded, with each nucleotide position on the + strand being represented by a complementary nucleotide on the - strand (that is, A pairs with T, G pairs with C).

When you sequence DNA, you have both the strands in your input, and both are amplified because PCR is not strand-specific.

However, you only want to read one strand at a time. Here's why. Sequencing starts at the 3' of the template. Let's assume you were looking at a 5-nucleotide long bit of DNA with the + strand sequence AATTG and the - strand sequence TTAAC. If both strands were read together, the read would be:

50% A 50% C
100% A
50% A 50% T
50% A 50% T
50% T 50% G

This means that you have no idea what the sequences are - the first nucleotide on the + strand could be an A, or a C, or the sequencing may have failed! To obtain a sequence that makes sense, it is necessary to look at them separately.

To do this, a short sequence is added to the ends of the double-stranded DNA. For example, if the 3' ligate were ggg, and the 5' were aaa, the sequences would become gggAATTG and TTAACaaa. Then, we use an adaptor that reads aaa - this cannot bind to the ligate in the second case, so only the + strand is read. Similarly, an adaptor of ggg would only allow the - strand to be read. Two runs, two sequences (which we expect to be complementary, and which we know the strand identity of) and no problems.