Figure 1: Template amplification strategies. | Nature Reviews Genetics

Figure 1: Template amplification strategies.

From: Coming of age: ten years of next-generation sequencing technologies

Figure 1

a | In emulsion PCR, fragmented DNA templates are ligated to adapter sequences and are captured in an aqueous droplet (micelle) along with a bead covered with complementary adapters, deoxynucleotides (dNTPs), primers and DNA polymerase. PCR is carried out within the micelle, covering each bead with thousands of copies of the same DNA sequence. b | In solid-phase bridge amplification, fragmented DNA is ligated to adapter sequences and bound to a primer immobilized on a solid support, such as a patterned flow cell. The free end can interact with other nearby primers, forming a bridge structure. PCR is used to create a second strand from the immobilized primers, and unbound DNA is removed. c | In solid-phase template walking154, fragmented DNA is ligated to adapters and bound to a complementary primer attached to a solid support. PCR is used to generate a second strand. The now double-stranded template is partially denatured, allowing the free end of the original template to drift and bind to another nearby primer sequence. Reverse primers are used to initiate strand displacement to generate additional free templates, each of which can bind to a new primer. d | In DNA nanoball generation, DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized and cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cleavage. This process is repeated for the remaining two adapters. The final product is a circular template with four adapters, each separated by a template sequence. Library molecules undergo a rolling circle amplification step, generating a large mass of concatamers called DNA nanoballs, which are then deposited on a flow cell. Parts a and b are adapted from Ref. 18, Nature Publishing Group.

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