Automatic DNA Sequencing Back

This facility has two automated capillary array based DNA Sequencers (Model 3730) from M/s Applied Biosystems, USA for analyzing fluorescently labeled DNA molecules. During the year, the facility was updated with another DNA Sequencer (Model 3730xl) for highthroughput applications. 

The systems are capable of sequencing 96/48 samples simultaneously, using 96/48 capillary arrays. Both sequencing and genotyping can be carried out in the same capillary arrays. All the models have an Argon ion laser excitation source, a CCD camera for detection of five fluorescent dyes simultaneously. Samples from 96/384 well plates are automatically loaded onto an array of 96/48 capillaries using robotics. More than 2300 sequencing samples can be processed in 24 hours with an average read length of 700 bases. The throughput for fragment analysis applications is roughly 38,000 genotypes per day. The different sizes of capillary arrays i.e. 36 & 50 cm provide read lengths ranging from 500 bp to 800 bp. The integrated built in Auto sampler, sample plate stacker and bar code reader provides full automation and easy sample tracking. 

Features like in capillary detection, dual side illumination and backside thinned CCD provide uniform signal and high optical sensitivity, resulting in accurate cost-effective analysis of DNA fragments. Software like Sequence analysis, Genotyper, SeqScape, Auto Assembler etc are also available in this facility. 

This facility also has a number of IBM and Macintosh computers networked together for downstream data analysis. Licensed versions of all the software are available on these systems and networked to the control computers for data download and analysis. 

DNA Sequencing: Clones and PCR products can be sequenced using Fluorescently-labeled Dye terminator cycle sequencing kits. Point mutation/base substitution, heterozygote and heteroplasmy (in case of mtDNA) can be detected by these systems using the supporting software. The sequencing facility has rendered a major contribution to some of the important projects concerning human genetic diversity, plants, animals, microorganisms, genetic disorders, etc. 

GeneScan analysis: Up to 16 PCR (STR) products can be analysed in a single capillary provided one of the PCR primer pairs is labeled with the fluorescent dyes. GeneScan analysis provides immense help in various disciplines, such as (a) Forensic medicine: Individual identification using STRs (b) Wildlife Forensic (c) Population studies: Study of genetic variability within and between populations using STRs (d) Medical applications: Identification of the repeat length of genetic disorders associated with trinucleotide repeat expansion (e) Development of DNA markers, particularly STRs for plants and animals (f) Amplified Fragment Length Polymorphism (AFLP): Used extensively for genetic mapping and characterization in plants and animals using fluorescent labeled primers.

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