3. Macroarray analysis – BESs and other molecular markers can be hybridized to
high-density BAC macroarrays to identify BACs that share molecular loci (i.e.,
overlapping BACs).
4. Polymerase chain reaction (PCR) – PCR screening of strategically pooled BACs
can be used as an alternative to macroarray analysis in identifying clones
containing shared loci.
5. Fluorescence in situ hybridization (FISH) – FISH can be used to visualize the
locations of markers/genes on chromosome preparations and position BACs with
respect to one another along chromosomes.
6. Optical mapping – Optical mapping is a relatively new physical mapping technique. In brief, DNA molecules are placed on a substrate containing microfluidic
channels. Single DNA molecules are stretched across the channels. The stretched
DNA molecules are treated with enzymes that either cut the DNA or nick the
DNA. In the former case, DNA-intercalating dyes can be used to visualize the
DNA molecules and the sites of restriction events – the fragment patterns of
the stretched DNA molecules can be compared (in a manner similar to DNA
fingerprinting) allowing overlapping pieces of DNA to be discovered. In the latter
case, the stretched DNA is treated with enzymes that make nicks at specific
sequences. The nicks are expanded via an exonuclease, and then a DNA polymerase is used to fill in the gap with fluorochrome-labeled nucleotides. The
pattern of fluorescence for a DNA molecule can be compared with other optically
mapped molecules to discover shared DNA regions (see, e.g., Lam et al. 2012).
BAC-based physical mapping (including DNA fingerprinting, BAC-end
sequencing, BAC macroarray screening or PCR-based BAC library screening, and
BAC-based FISH) was traditionally used as a means of positioning large pieces of
DNA with respect to one another to obtain a minimum tiling path, i.e., the minimum
number of clones collectively representing all, or an extensive part of, a chromosome. Sequencing the clones in a chromosome’s minimum tiling path served as a
means to produce a high-quality chromosome sequence (see Peterson 2014 for
review).
3.6 emPCR and Bead Colonies
The sequencing techniques of 454, SOLiD, and Ion Torrent amplify DNA by using
beads containing millions of identical capture sequences. Genomic DNA (target
DNA) with adapters ligated to its ends is denatured and mixed with an excess
quantity of beads. One of the adapters on each target molecule is complementary
to the capture probes on the beads. The low amount of DNA compared to beads
ensures that, on average, no bead will have more than one DNA sequence hybridize
to one of its capture probe sequences. PCR reagents and oil are added to the beads,
and the mixture is agitated to create an emulsion. The beads are of a size that
promotes the formation of an aqueous layer (including the PCR reagents) around
Sequencing Plant Genomes
125
high-density BAC macroarrays to identify BACs that share molecular loci (i.e.,
overlapping BACs).
4. Polymerase chain reaction (PCR) – PCR screening of strategically pooled BACs
can be used as an alternative to macroarray analysis in identifying clones
containing shared loci.
5. Fluorescence in situ hybridization (FISH) – FISH can be used to visualize the
locations of markers/genes on chromosome preparations and position BACs with
respect to one another along chromosomes.
6. Optical mapping – Optical mapping is a relatively new physical mapping technique. In brief, DNA molecules are placed on a substrate containing microfluidic
channels. Single DNA molecules are stretched across the channels. The stretched
DNA molecules are treated with enzymes that either cut the DNA or nick the
DNA. In the former case, DNA-intercalating dyes can be used to visualize the
DNA molecules and the sites of restriction events – the fragment patterns of
the stretched DNA molecules can be compared (in a manner similar to DNA
fingerprinting) allowing overlapping pieces of DNA to be discovered. In the latter
case, the stretched DNA is treated with enzymes that make nicks at specific
sequences. The nicks are expanded via an exonuclease, and then a DNA polymerase is used to fill in the gap with fluorochrome-labeled nucleotides. The
pattern of fluorescence for a DNA molecule can be compared with other optically
mapped molecules to discover shared DNA regions (see, e.g., Lam et al. 2012).
BAC-based physical mapping (including DNA fingerprinting, BAC-end
sequencing, BAC macroarray screening or PCR-based BAC library screening, and
BAC-based FISH) was traditionally used as a means of positioning large pieces of
DNA with respect to one another to obtain a minimum tiling path, i.e., the minimum
number of clones collectively representing all, or an extensive part of, a chromosome. Sequencing the clones in a chromosome’s minimum tiling path served as a
means to produce a high-quality chromosome sequence (see Peterson 2014 for
review).
3.6 emPCR and Bead Colonies
The sequencing techniques of 454, SOLiD, and Ion Torrent amplify DNA by using
beads containing millions of identical capture sequences. Genomic DNA (target
DNA) with adapters ligated to its ends is denatured and mixed with an excess
quantity of beads. One of the adapters on each target molecule is complementary
to the capture probes on the beads. The low amount of DNA compared to beads
ensures that, on average, no bead will have more than one DNA sequence hybridize
to one of its capture probe sequences. PCR reagents and oil are added to the beads,
and the mixture is agitated to create an emulsion. The beads are of a size that
promotes the formation of an aqueous layer (including the PCR reagents) around
Sequencing Plant Genomes
125