2.6 years ago by
Spain. Universidad de Córdoba
You break your DNA or cDNA into million/billions of different and random pieces.
- Then, you add adapters
- Then you add to your flow cell all of these random pieces. Each piece will land in a different part of the flow cell
- So at this time, you only have in each place where the DNA has landed a single chain of DNA.
- If you would add your reagents (read labeled precursors of the DNA) at this point to ascertain the DNA sequence, only ONE chain of the DNA will be used to sequence that particular DNA. That means that only ONE labeled precursor will be added for each of the DNA fragments
- And this means that you will get a very poor signal, as only one labeled precursor will be measured in each cycle
- To overcome this, every single and individual piece of DNA, already stuck to the flow cell, are PCR amplified. In Illumina, this PCR amplification is done ina a way that you generate a cluster of DNA fragments coming from a single DNA piece to form hundreds of clonal fragments of DNA, each located at the same place
- Thus, you will be able to measure the simultaneous incorporation of many labeled precursors at the same time, then making feasible for the device to measure it