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DNA Sequencing facility


 

Facility Overview  

The HIV/AIDS Department has a centralized DNA sequencing facility which has been used extensively by researchers working in the fields of HIV, TB and pharmacogenetics. This facility has a brand new AB 3500 Avant Genetic Analyzer eight capillary DNA sequencer (Applied Biosystems).

Specifications  

The AB 3500 Genetic Analyzer is a multi-capillary electrophoresis instrument designed to separate amplified DNA product based on size and record the resulting data in a computerized data file capable of being analyzed using specialized software.

Working principle

 

Capillary electrophoresis separates DNA fragments based on their size to charge ratio. The cathode is placed into the sample. An electrical pulse activates the migration and separation of the DNA through the capillary. The negatively charged DNA migrates from the cathode to the anode because of the attraction of opposite charges. Smaller DNA fragments migrate faster than larger fragments thus reaching the detector sooner. The DNA fragments have fluorescently-labelled primers attached so that when the DNA goes past the detection window, a narrow beam of light from the laser excites the dyes. The excitation of the dyes give off an emission wavelength which is a longer wavelength of light than the laser's excitation wavelength in all directions. Some of this light passes through a diffraction grating, which then sends the light to the CCD detector. The CCD detector can detect the colour based on the wavelength. Along with an internal size standard and allelic ladder, a software program takes these peaks that are detected and give it a specific allele designation for a given locus. The combinations of all of the fluorescent peaks give rise to an electropherogram.


APPLIED BIOSYSTEMS 3500 GENETIC ANALYZER

Applications

 

The 8-capillary 3500 DNA analyzer can be used for traditional DNA sequencing and DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis and mutation detection.

 

 

Service

Sequencing application charges

 
University
National Lab/R&D's
Sequencing of Purified PCR products
(single primer provided by user)
Rs. 350
Rs. 700
Sequencing of un-purified PCR products
(single primer provided by the user)
Rs. 500
Rs. 900

 

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