Overview of DArT technology
DArT technology consists of several steps:
- Complexity reduction of the DNA of interest
- Library creation
- Microarraying libraries onto glass slides
- Hybridisation of fluoro-labelled DNA onto slides
- Scanning of slides for hybridisation signal
- Data extraction and analysis
Complexity reduction
DArT works by reducing the complexity of a DNA sample to obtain a 'representation' of that sample. Our preferred method of complexity reduction relies on a combination of restriction enzyme digestion and adapter ligation, followed by amplification (Wenzl et al., 2004). However, an infinite range of alternative methods can be used to prepare genomic representations for DArT.
DArT operates on the principle that the genomic 'representation' contains two types of fragments:
- Constant fragments, found in any 'representation' prepared from a DNA sample from an individual belonging to a given species, and
- Variable (polymorphic) fragments called molecular markers, only found in some but not all of the 'representations'.
The variable fragments are informative because they reflect sequence variation that determines the fraction of the original DNA sample that is included in the 'representation'. We call the variable fragments DArT markers. Their presence vs. absence in a genomic 'representation' is assayed by hybridising the 'representation' to a DArT array consisting of a library of that species.
Library creation
To create a library for any species, a mixture of genomic 'representations' from a pool of individuals covering the genetic diversity of the species is amplified. These fragments are cloned into a vector that is introduced into E. coli to form a library. Within the library, each colony contains one of the fragments from the genomic 'representation'.
Microarraying
At present the high-throughput capability of DArT is based on a microarray platform. After library creation, a selection of clones from the library are arranged into a plate format (usually 384-well plates). The fragments within the library are amplified and spotted onto glass slides using a microarrayer to form a genotyping array.
Hybridisation
The genotyping arrays are hybridised with genomic 'representations' of individual DNA samples prepared using the same complexity reduction method. These individual 'representations' are labelled with one fluorescent label, while the vector fragment is labelled with another fluorescent label to act as a reference. Each individual 'representation' will only hybridise to matching fragments on the genotyping array, thereby displaying a unique hybridisation pattern.
Scanning
The hybridised slides are first washed and processed to remove unbound labelled DNA. The slides are then scanned using a scanner to detect fluorescent signal emitted from the hybridised fragments. The result from each fluorescent channel is recorded and the resulting images are stored in tif format.
Data analysis
The data from the scanned images is extracted and analysed using the DArTsoft software and the information is managed by the DArTdb Laboratory Information Management System.
