Материал из Zbio
All second-generation platforms (except single-molecular methods) rely on some clonal amplification method.
[править] Bridge amplification (two-dimensional PCR) / Illumina
Two-dimensional PCR performed is on Cluster station (Flowcell preparation) and on Genome Analyser (regeneration of clusters for Paired-end sequencing). Here is a scheme for Cluster station amplification.
[править] Library deposition
[править] Surface amplification
Opposite to conventional PCR, surface amplification is performed on constant temperature (60°C). Formamide works as a denaturing agent. Normally, 35 cycles of pumping:
- formamide (at 60°C formamide melts DNA duplexes; equivalent to "denaturation step" in normal PCR);
- extension buffer (non-denaturating conditions, equivalent to "anealing step" in normal PCR);
- extension mixture (primer extension step, equivalent to "extension step" in normal PCR).
In contrast to normal PCR all solutions are used only for one cycle and then go to the trash. It helps to keep low background. Otherwise occasionally detached DNA molecule could have a chance to hybridize in another place and start a new cluster there. Surface PCR has lower efficiency if compare with PCR in solution. 35 cycles result in ~1000 copies of the original molecule (in ideal PCR 35 cycles should give ~1010x amplification). The possible reasons of lower efficiency:
- DNA duplex has high chances to renature again instead of hybridising with a new PCR primer;
- cluster has no free PCR primers in the center and grows only on periphery;
Original library molecule is denatured and washed away.
Extended molecule bends and hybridizes to a second PCR primer (forms a bridge).
Extension of hybridized primer.
Two DNA strands are denatured.
Extended molecules may hybridize to each other again or find other PCR primers.
In the second case they will duplicate again.
DNA strands are denatured.
[править] Flowcell processing
After clonal amplification clusters should be converted into single-stranded form and prepared for sequencing.
-- blocking of all 3' ends (ddNTP's and terminal transferase) to prevent extension of DNA molecules on each other;
-- annealing of sequencing primer.
[править] Emulsion PCR (ePCR) / SOLiD, 454
Emulsion may be considered as a large number of independent PCR reactions.
|1, 4, 6, 9, 10, 12||-||-||no template: no PCR, no matter if paramagnetic bead is present or not. Paramagnetic beads which do not participate in amplification may be easily separated on "enrichment" step|
|5||+||-||one template molecule in the drop: clonal PCR reaction, but there is no bead in the drop|
|13||+||-||P2-P2 adaptors: amplified molecules have no chance to stick to the bead|
|8, 11||+||+||ideal situation: one template molecule and one bead in a drop: clonal amplification, one bead generated from each clone|
|3||+||+||acceptable situation: one template and two beads. Two beads with the same template will be generated|
|7||+||+||two different template molecules. Bead will be covered by a mixture of different DNA molecules and will come through the enrichment procedure. Sequencing will fail|
|2||+||+||one double-stranded DNA molecule is a heterohybrid of different clones (as a result of template annealing during preamplification). Bead will be covered by a mixture of different DNA molecules and will come through the enrichment procedure. Sequencing will fail|