each bead, while the hydrophobic oil keeps beads separated from one another.
Emulsion PCR (emPCR) is performed so that each bead to which a target molecule
had hybridized will end up with thousands of identical DNA molecules attached to
its surface. The emulsion is then broken, and only those DNA strands synthesized
via the elongation of capture probes remain attached to the beads. In 454 and Ion
Torrent sequencing, the beads are deposited into the wells of a microfabricated plate.
In contrast, in SOLiD, the beads are fixed onto a glass slide (Heather and Chain
2016).
There is no official name for bead-based amplification of DNA molecules. We
will refer to a bead attached to clonally amplified molecules as a “bead colony.”
3.7 Bridge Amplification and Clusters
Illumina does not use emPCR to create groups of amplified molecules. Instead,
target molecules flanked by adapters are hybridized to adapter complementary
sequences fixed to lanes of a glass slide. A process known as bridge amplification
is used to generate DNA colonies of amplicons (see Fig. 5). The resulting DNA
colonies/spots are called clusters.
5
3.8 First-Generation Sequencing
The first sequencing of nucleic acids was performed in the 1960s using short, readily
available tRNA and rRNA molecules as substrates. The general approach, which
was later adapted to sequence small viral genomes, involved full and partial restriction digests of a nucleic acid molecule of interest coupled with fractionation of the
resulting fragments. These restriction enzyme-based strategies were highly limited in
scope and not readily utilized except in a few laboratories (Heather and Chain 2016).
In 1977 two papers describing new DNA sequencing techniques were published
back-to-back in the Proceedings of the National Academy of Sciences of the United
States of America (PNAS). Unlike the methods used before, these two methods
could, in theory, be used to sequence any DNA molecule. The first paper described a
technique, now known as Maxam-Gilbert sequencing, which involved isolation of a
restriction fragment band containing thousands of copies of the same DNA molecule; radiolabeling of fragments at their 5
0 ends; separation of the DNA strands by
denaturing electrophoresis; selection of one of the strands for sequencing; distribution of the molecule to be sequenced into four separate tubes; chemical treatment of
the contents of each tube with an agent(s) that, at low frequency, preferentially
5 emPCR with beads and bridge amplification are not the only means of producing DNA polymerase
colonies (see Fuller et al. 2009 for review).
126
D. G. Peterson and M. Arick
Emulsion PCR (emPCR) is performed so that each bead to which a target molecule
had hybridized will end up with thousands of identical DNA molecules attached to
its surface. The emulsion is then broken, and only those DNA strands synthesized
via the elongation of capture probes remain attached to the beads. In 454 and Ion
Torrent sequencing, the beads are deposited into the wells of a microfabricated plate.
In contrast, in SOLiD, the beads are fixed onto a glass slide (Heather and Chain
2016).
There is no official name for bead-based amplification of DNA molecules. We
will refer to a bead attached to clonally amplified molecules as a “bead colony.”
3.7 Bridge Amplification and Clusters
Illumina does not use emPCR to create groups of amplified molecules. Instead,
target molecules flanked by adapters are hybridized to adapter complementary
sequences fixed to lanes of a glass slide. A process known as bridge amplification
is used to generate DNA colonies of amplicons (see Fig. 5). The resulting DNA
colonies/spots are called clusters.
5
3.8 First-Generation Sequencing
The first sequencing of nucleic acids was performed in the 1960s using short, readily
available tRNA and rRNA molecules as substrates. The general approach, which
was later adapted to sequence small viral genomes, involved full and partial restriction digests of a nucleic acid molecule of interest coupled with fractionation of the
resulting fragments. These restriction enzyme-based strategies were highly limited in
scope and not readily utilized except in a few laboratories (Heather and Chain 2016).
In 1977 two papers describing new DNA sequencing techniques were published
back-to-back in the Proceedings of the National Academy of Sciences of the United
States of America (PNAS). Unlike the methods used before, these two methods
could, in theory, be used to sequence any DNA molecule. The first paper described a
technique, now known as Maxam-Gilbert sequencing, which involved isolation of a
restriction fragment band containing thousands of copies of the same DNA molecule; radiolabeling of fragments at their 5
0 ends; separation of the DNA strands by
denaturing electrophoresis; selection of one of the strands for sequencing; distribution of the molecule to be sequenced into four separate tubes; chemical treatment of
the contents of each tube with an agent(s) that, at low frequency, preferentially
5 emPCR with beads and bridge amplification are not the only means of producing DNA polymerase
colonies (see Fuller et al. 2009 for review).
126
D. G. Peterson and M. Arick