Optical mapping

'Optical mapping'is a technique for constructing ordered, genome-wide, high-resolution restriction maps from single, stained molecules of DNA, called "optical maps". By mapping the location of restriction enzyme sites along the unknown DNA of an organism, the spectrum of resulting DNA fragments collectively serve as a unique "fingerprint" or "barcode" for that sequence.

"Ordered Restriction Maps of Saccharomyces Cerevisiae Chromosomes Constructed by Optical Mapping." 

Technology

1. Genomic DNA is obtained from lysed cells, and randomly sheared to produce a "library" of large genomic molecules for optical mapping.
2. A single molecule of DNA is stretched (or elongated) and held in place on a slide under a fluorescent microscope due to charge interactions.
3. DNA molecule is digested by added restriction enzymes, which cleave at specific digestion sites. The resulting molecule fragment remain attached to the surface. The fragment ends at the cleavage site are drawn back (due to elasticity of linearized DNA), leaving gaps which are identifiable under the microscope as gaps.
4. DNA fragments stained with intercalating dye is visualized by fluorescence microscopy and are sized by measuring the integrated fluorescence intensity. This produces an optical map of single molecules.
5. Individual optical maps are combined to produce a consensus, genomic optical map.

History of Optical Mapping Platform

Early system

DNA molecules were fixed on molten agarose developed between a cover slip and a microscope slide. Restriction enzyme was pre-mixed with the molten agarose before DNA placement and cleavage was triggered by addition of magnesium.

Using charged surfaces

Rather than being immobilized within a gel matrix, DNA molecules were held in place by electrostatic interactions on a positively-charged surface. Resolution improved such that fragments from ~30kb to as small as 800bp could sized.

Automated system

This involved the development and integration of an automated spotting system to spot multiple single molecules on a slide (like a microarray) for parallel enzymatic processing, automated floresence microscopy for image acquisition, image procession vision to handle images, algorithms for optical map construction, cluster computing for processing large amounts of data

High-throughput system using microfluidics

Observing that microarrays spotted with single molecules did not work well for large genomic DNA molecules, microfluidic devices using soft lithography possessing a series of parallel microchannels was developed.

Next-generation system using nanocoding technology

An improvement on optical mapping, called "Nanocoding", has potential to boost throughput by trapping elongated DNA molecules in nanoconfinements.

Other genome analysis methods

There are a variety of approaches to identifying large-scale genomic variations (such as indels, duplications, inversions, translocations) between genomes. Other categories of methods include using microarrays, pulsed-field gel electrophoresis, cytogenetics and paired-end tags.

Uses

Initially, the optical mapping system has been used to construct whole-genome restriction maps of bacteria, parasites, and fungi.

Optical Sequencing

Optical sequencing is a single molecule DNA sequencing technique that follows sequence-by-synthesis and uses optical mapping technology.

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Optimization Strategies

Selection of an appropriate DNA polymerase is critical to the efficiency of the base addition step and must meet several criteria:

• Ability to efficiently incorporate FdNTP at consecutive positions
• Lack of 3'-5' exonuclease and proofreading activity to prevent the removal newly incorporated FdNTP
• High fidelity to minimize mis-incorporations
• Good activity on templates which are mounted to surfaces (eg. optical mapping surface)

Advantages

Single-molecule analysis
Since minimal DNA sample required, time-consuming and costly amplification step is avoided to streamline sample preparation process.

Large DNA molecule templates (~500 kb) vs. Short DNA molecule templates (< 1kb) While most next generation sequencing technologies aim of massive amounts of smalls sequence reads, these small sequence reads make de novo sequencing efforts and genome repeat regions difficult to comprehend. Optical sequencing uses large DNA molecule templates (~500 kb) for sequencing and these offers several advantages over small templates:

Disadvantages

• Single molecule DNA sequencing requires a high level of precision to match the confidence from the redundant read coverage provided by current next-generation sequencing technologies.
• Nicks on both strands at similar positions resulting in low template during sequence-by-synthesis.
• Fluorochrome-labeled nucleotides are not removed after incorporation and because of these bulky labels, multiple incorporation might be difficult.

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