Tutorial

DNA can be extracted from whole blood using a standard phenol/chloroform extraction method. A battery of 25 microsatellite markers which are based on the guidelines of ISAG & FAO’s DAD-IS program  can be utilised to  generate allelic data. Only forward primers at 5' end of each pair needs labelling with one of the four fluorophores, i.e., FAM (blue), VIC (green), NED (yellow) and PET (red). For details please see Kumar et al., 2009. PCR reactions may be carried out using  50–100 ng genomic DNA in a 25 µl reaction volume.

A touchdown PCR protocol can be used with initial denaturation of 95◦C for 1 min, 3 cycles of 95◦C for 45 s, and 60◦C for 1 min, 3 cycles of 95◦C for 45 s, and 57◦C for 1 min, 3 cycles of 95◦C for 45 s, and 54◦C for 1 min, 3 cycles of 95◦C for 45 s and 51◦C for 1 min, 20 cycles of 95◦C for 45 s, and 48◦C for 1 min with final extension at 72◦C for 5 min. At the end   reaction can be stopped by adding  5.0 µl of stop dye (95% formamide, 0.25% bromophenol blue and 0.25% xylene cyanol). 6 µl of PCR products  can be loaded on a 2% agarose gel,  for electrophoresis to visualize it in  UV light after ethidium bromide staining to detect the amplification.

The PCR products can be mixed in a ratio of 1:1.5:2:2 of FAM, VIC, NED and PET labeled primers, respectively. 0.5 µl of this mixture (a post PCR multiplex of 4–5 primers) can be combined with 0.3 µl of Liz 500 as internal lane standard  of Applied Biosystems, U.S.A.  and 9.20 µl of Hi-Di Formamide per sample. The resulting mixture should be  denatured by incubation for 5 min at 95◦C. These denatured samples has to be  run on automated DNA sequencer of Applied Biosystems (ABI 3100 Avant). The electropherograms drawn through Gene Scan can be used to extract DNA fragment sizing details using Gene Mapper software (version 3.0) (Applied Biosystems, U.S.A.).

Schematic Diagram

 

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How to use the GOMI Web server

The purpose of GOMI is to predict the Goat breed based on DNA Marker data. The Homepage has seven different tabs viz. Home, Submission, Algorithm, Tutorial, Team, Links, FAQs.

To use the GOMI Web-server to predict the goat breed, Submission tab needs to be used. Algorithm tab gives the details of the algorithm used for the development of this web-server.

Tutorial page, Links, and FAQs are also available for assisting the users.

The diagrammatical Illustration of how to use the GOMI server is as follows:

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Input Methods :

Data can be input either by manually entering the data in the form or by uploading a .txt or .csv file with 50 values.

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The result gives the probability of a particular breed or breeds in the DNA marher data provided.

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