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Clonal amplification

Bridge amplification Emulsion PCR Rolling circle amplification  Comments

All second-generation platforms (except Helicos) rely on some clonal amplification method.




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.





Library deposition

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


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).


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




Surface amplificationa

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 trash. It helps to keep low background. Othervise 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 renaturate again instead of hybridising with a new PCR primer;
• cluster has no free PCR primers in the center and grows only on perifery;

Formamide
Original library molecule is denatured and washed away.


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


Extension mixture
Extension of hybridized primer.


Formamide washing
Two DNA strands are denatured.



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


Extension mixture
In the second case they will duplicate again.


Formamide washing
DNA strands are denatured.



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


After 35 cycles cluster consists on a number of double-stranded bridges.







Flowcell processing

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

DNA is denaturated.



One primer is cut off and washed out.



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.








Emulsion PCR (ePCR) / SOLiD, 454



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 solition 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.







drops result amplification PMP-binding description 1, 4, 6, 9, 10, 12 acceptable - - 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 good + + 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 bad + + 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









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.