Automated DNA Sequencing

ABI PRISM 3100 Genetic Analyzer

The Genomics Core Facility uses two ABI PRISM 3130XL Genetic Analyzers. These are multi - color fluorescence based DNA analysis system that utilizes the proven technology of capillary electrophoresis with 16 capillaries operating in parallel. This instrument uses a laser to excite fluorescently labeled DNA fragments as they pass the laser window moving down a the capillary array. The fluorescence is detected and signals are sent to a computer and collected by the ABI Data Collection Software. This data is then processed and interpreted by the ABI Sequence Analysis software. The final output is a color profile or a chromatograph which displays peaks and corresponding base calls. Peaks are four different colors, one for each base.

Automated DNA Sequencing Guidelines

Order Form

Downloading Sequencing Results

Quantitative real-time PCR (qr-PCR)

The ABI PPRISM 7900HT is an integrated system that detects and quantitates nucleic acid sequences using fluorescent primers and probes (Taqman) or Syber green.  This high-throughput real-time PCR system is designed to perform both real-time PCR and post-PCR (end-point) analysis.  It is designed to accept user interchangeable 96-well, 384-well and Low Density Array Card sample blocks, allowing for greater flexibility. This instrument also comes with an automation accessory capable of loading and unloading reaction plates. The resulting integrated unit should provide unattended instrument operation for the processing of up to 84 384-well reaction plates.  This would allow for unattended processing of at least 5,000 real-time quantitative PCR sample wells per day (24 hour period), and at least 10,000 SNP genotyping samples every hour.

Key applications that can be performed on the ABI PRISM 7900HT included Viral quantitation, quantitation of Gene expression, array verification, drug therapy efficacy, DNA damage measurement, quantity control and assay validation, pathogen detection, and genotyping.  Whatever the application, qr-PCR is based on the 5’ nuclease assay (TaqMan).  Briefly, this assay includes, a pair of un-labeled primers (amplify target gene of interest) and a duel labeled primer (reporter and quencher dye).  During PCR amplification, the probe which anneals to a specific complementary sequence between the forward and reverse primer is displaced and gets cleave by the 5’-3’ exonuclease activity of Taq.  Once cleaved, the two dyes are not longer in close proximity, which allows emission of the reporter dye.  A website: http://cgr.otago.ac.nz/SLIDES/TAQMAN/SLD010.HTM  gives a nice overview of this procedure.

USEFUL WEBSITES:

1. Essentials of Real Time or "Kinetic" PCR

https://docs.appliedbiosystems.com/pebiodocs/00105622.pdf

2. Relative Quantitation of Gene Expression

http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf

3. Multiplex PCR with TaqMan VIC probes

http://docs.appliedbiosystems.com/pebiodocs/04306236.pdf

4. To order Taqman primers and probes

http://www.appliedbiosystems.com/products/productdetail.cfm?prod_id=92

5. Pre-designed human gene expression assays:

gene expression assays on demand

6. Pre-designed human SNP assays:

snp assays on demand

7. Assays by design service (any organism):

http://www.appliedbiosystems.com/products/productdetail.cfm?prod_id=761

Beckman Biomek 2000 Robot

The Beckman Biomek is a liquid handling robot. The Biomek 2000 can do anything you can do with a pipetman, but it can do it continuously, more accurately, and without mistakes (assuming it is programmed correctly). For example, it can make up a PCR master mix, and set up the thermal cycling reactions in a 384-well plate that has a different template in each well. The Biomek 2000 has 12 positions on its deck for holding tips, as well as a variety of plates, tubes, and fluid reservoirs. The robot has the following pipetors: continual flow (for washing), P1000, P250, P20, P250 8-channel, P20 8-channel.

Beckman Biomek 2000 Robot

The Biomek 2000 Laboratory Automation Workstation meets the demands of rapidly changing life science technology with simple, intelligent automation of liquid-handling tasks. Pipetting, diluting and dispensing operations are performed quickly, easily and automatically. The modular platform allows expansion of system capability to include plate heating and cooling, plate washing, high-density transfers, photometric measurement and high-capacity operation. The entire system is controlled by powerful and user-friendly BioWorks software with an intuitive graphical interface.

As this equipment is very expensive and easy to damage, the facility is requiring all potential users to be trained by Mrs. Flynn or by a factory representative.

Genotyping (Coming Soon)

Genome Scan

Applied Biosystems MD10 Linkage Mapping Set. This commercial set is a group of 400 di-nucleotide markers covering the somatic and X chromosomes at an average of 10cM. Panels are arranged by chromosome, facilitating partial genome scans. The electropherogram below is a single panel of the MD10 Linkage Mapping Set composed of 17 markers.

Single Nucleotide Extension (SNE)

Applied Biosystems SNaPshot kit for single nucleotide extension reactions. This is essentially a "mini-sequencing" reaction using non-fluor primers and the same Big Dye terminators used in fluorescent sequencing. The multi-step process involves 1) PCR of a region containing a known SNP, 2) ExoSap clean-up of excess dNTPs and primers, 3) SNE reaction using an interrogation primer and dye labeled ddNTP's, 4) Sap cleanup and 5) Gel electrophoresis.

The result is a single color peak for each specific nucleotide incorporated. The electropherogram below shows six multiplexed SNE reactions.