Clonal amplification

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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

Bridge amplification -- surface with primers
Two PCR primers are attached to the surface of flowcell. One of the primers has a cleavable site (cross on red primer).


Bridge amplification -- annealing pf the library molecule
Preamplified library is denaturated in NaOH, then hybridization buffer is added to shift pH to neutral value. Library is loaded into the channel in neutral aquatious solution. DNA molecules can hybridize to the PCR primers. Red primer hybridize with a library molecule on the picture; complementary strand of this molecule have a chance to form a hybrid with a green primer.


Bridge amplification -- copying of library molecule
Elongation reaction: extension mix (buffer, dNTP's, Taq polymerase) is pumped into a channel. Hybridized primer extended on library molecule.


[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;


Bridge amplification -- denaturation
Formamide
Original library molecule is denatured and washed away.


Bridge amplification -- annealing
Extension buffer
Extended molecule bends and hybridizes to a second PCR primer (forms a bridge).


Bridge amplification -- extension

Extension mixture
Extension of hybridized primer.


Bridge amplification -- denaturation
Formamide washing
Two DNA strands are denatured.



Extension buffer
Extended molecules may hybridize to each other again or find other PCR primers.

Bridge amplification -- renaturation

Bridge amplification -- annealing to new primers



Bridge amplification -- extension
Extension
In the second case they will duplicate again.


Bridge amplification -- denaturation
Formamide washing
DNA strands are denatured.


.............................


Bridge amplification -- ready double-stranded clusters
After 35 cycles cluster consists on a number of double-stranded bridges.

[править] Flowcell processing

After clonal amplification clusters should be converted into single-stranded form and prepared for sequencing.


Bridge amplification -- denaturation of clusters
DNA is denaturated.


Bridge amplification -- denaturation of clusters
One primer is cut off and washed out.


Bridge amplification -- blocking
the last operations, which are done on Cluster station are:

-- 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

Scheme of emulsion PCR
Amplification reaction is performed in water-in-oil emulsion in the presence of low concentration of library molecules and paramagnetic particles coated by one PCR primer. This PCR primer is also present in the solution but in low concentration: it is enough for early cycles of PCR, but then primers on beads should be involved into amplification.

Emulsion may be considered as a large number of independent PCR reactions.


dropsamplificationPMP-bindingdescription
acceptable result
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
good result
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
bad result
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

[править] Rolling circle amplification

Scheme of rolling circle amplification
Rolling circle amplification: φ29 DNA polymerase is capable to make up to 103 single-stranded copies of original circle molecule. The multiplication number is significantly different for different templates. So, it is necessary to select only large molecules for sequencing.


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