Frequent Asked Questions
The DNA needs to be suitable for digestion by restriction enzymes and ligation to adaptors.
Although additional preparation is required, the investment in higher purity DNA has the following long term advantages:
- improved shipping endurance;
- improved storage duration;
- higher data quality and volume; and
- broader usage over multiple tests and assays, over a longer time period.
We recommend using the following protocol prior to sending the samples to us. This will help you receive the best quality data for your samples in less time, preventing you from having to resend those samples of poor quality if any.
DNA quality of the samples is controlled by:
Incubating 1 ul of DNA in Restriction Enzyme buffer at 37°C for 2 hours.
Resolve the DNA on a 0.8% agarose gel in 1x TAE buffer.
Inspect the gel image for your samples. Good quality DNA gives a high molecular weight band on the gel image.
This protocol is a mock digest to detect the presence of buffer-activated (usually Mg dependent) nucleases: no restriction enzyme is used.
Another option for you is to send us the image of the DNA quality control you already have for us to ensure the DNA is of sufficient quality for us to proceed with genotyping.
To perform a DArT assay, we require that you send us about 10-20 µl of an aqueous solution of DNA at 50 to 100 ng per µl, not more. Excessive concentration is detrimental to the assay. If your DNA is concentrated: dilute it.
The assay itself requires less than one µl but we need a larger volume to reduce problems during shipment and to be able to perform Quality Control.
DArT markers are sequence-ready and can be quickly sequenced when the need arises.
For Barley, Oats, Sorghum, Sugarcane, Cucumber and Wheat, most markers have been sequenced.
We are nearing completion of sequencing apple markers (approximately 3,000), eucalyptus (>7,000) and progressing with sequencing markers from many other species (e.g. potato, lolium, festuca, cassava, triticale, cotton, rye etc.) Prior to their publication, sequences are available upon request.
For other species, sequencing will be organised as demand for the data increases.
You would save time and money if you check your DNA before shipping to us. We suggest that you follow the QC procedure we use, testing for the presence and quantity of DNA as well as the lack of nucleases, on an agarose gel.
You may want to control further the suitability of the DNA for DArT, by testing the digestibility of the DNA using restriction enzyme PstI and/or a frequently cutting enzyme, always running side by side cut and uncut DNA, in the same buffer.
A complete DArT assay from QC-passed DNA to data can take as little as 3 days.
Of course we try to service many users at the same time, so our genotyping service aims at delivering data to the customers within 8 weeks of receiving suitable quality DNA.
Our preferred methods of payment are via:
- Direct fund transfer to our Australian accounts (The invoice number MUST be quoted in the reference or message section of the transfer form);
- Credit card - Visa, MasterCard, American Express, Diners, JCB (Note: A 3.5% surcharge applies); or
- Cheque (Made payable to Diversity Arrays Technology Pty Ltd).
Services are invoiced on your receipt of the data, however invoicing in advance and quotes are also available.
Please refer to the:
"OUR REQUIREMENTS" and
sections in the sample service specification [PDF].
DArT markers can be used in Marker Assisted Selection (reference). Using the sequence of the DArT marker of interest, you will need to establish a monoplex assay. This will usually require the:
- Sequencing of the genomic region of interest;
- Identification of an appropriate sequence polymorphism in that region; and
- Design of an assay (nearly always a PCR-based assay) to type this polymorphism.
Before you embark into the significant work required to establish one or a few monoplex assays, carefully consider the density of information you require.
We can provide low to medium density (i.e. 100-1,000 markers) assays customised to your application.
The low-density assay can type a small number of genomic regions of interest together with some "genetic background" markers.