08. Bridge PCR

Bridge PCR is a PCR technique that embeds DNA on a surface for cloning. It is used by Illumina's HiSeq platform.

Procedure

1) Adapted attached to ends of fragmented DNA

The DNA is fragmented (through sonication or any other method) and adapters are ligated to both ends.

Adapters being attached to fragmented DNA.
Adapters are attached to fragmented DNA.

2) Denature and bind to flow cell surface

The DNA is then denatured into single-stranded molecules. These fragments are then floated onto a flow cell which have corresponding adapter sequences that permit binding.

When the DNA strands are placed onto the slide, they attach to their corresponding adapter sequences.

DNA strands are ligated onto the flowcell.
DNA strands are ligated onto the flowcell.

3) Bridge amplification

Add dNTPs, and DNA polymerase enzyme to elongate DNA strands.

Bridge Amplification
Bridge amplification occurs with regular PCR components.

4) Denature and repeat to generate clusters

Denaturation the newly formed DNA strands and repeat until dense clusters of dsDNA are generated in each channel of flow cell.

Bridge Amplification final step.
Repeat until we have millions of dense clusters of DNA.

The reverse strands are then cleaved and washed away.

What is the purpose of high density regions?

After Bridge PCR is conducted on the flow cell, Illumina uses fluorescently labeled dNTP's to detect each nucleotide bases.

So for example, if a red fluorescent light goes off, then we know it's an A. If a blue light goes off, then we know it's a G, and so forth (colors here aren't accurate but you get the picture).

However, the signal produced by the synthesis of one dNTP on a strands is not enough to be detected. This is why we need to amplify the DNA sequences and producing a dense amount of sequences per area on the flow cell.

We'll see how Illumina is able to sequence millions of these dense colonies in parallel.

References

Illumina Website

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