Fluorescence-based DNA sequencing systems use dideoxy chain termination reactions to generate labeled reaction products that are size-fractionated by gel electrophoresis. A one-sided PCR amplification strategy, called cycle sequencing, that utilizes a modified Taq DNA polymerase and temperature cycling is used in automated DNA sequencing to generate high levels of chain terminated product from a small amount of template. The use of cycle sequencing has greatly extended the automation capabilities of DNA sequencing because it requires much less starting material for each round of sequencing.

The Applied Biosystems Incorporated (ABI) system uses four spectrally distinct fluorescent ddNTPs and a proprietary Taq polymerase called AmpliTaq. This strategy makes it possible to perform the entire sequencing reaction in a single tube and to resolve the chain-terminated products in one lane of a sequencing gel. The ABI DNA detection system permits direct real time data acquisition by laser-activated dye excitation at a point in the electrophoresis run that maximizes fragment resolution.

Fluorochrome excitation and emission detection occur at a fixed point near the bottom of the gel to permit maximum resolution. Sequence data are recovered as an emission spectra readout for each fluorescent dye as a function of fragment size. A text file of the inferred DNA sequence is output directly to an in-line computer.

The ABI PRISM® 3700 DNA Analyzer is an electrophoresis system developed specifically for production-scale DNA analysis. To meet the requirements of the production laboratory, the system provides walkaway automation (24-hour unattended operation), low operating costs, sensitivity and reliability. PE Corporation has developed a new generation of fluorescent dyes called dRhodamine for use with the ABI 3700 machine.

The University of Arizona DNA Sequencing Core Service is managed by the Laboratory of Molecular and Systematics Evolution (LMSE) as part of the Arizona Research Labs. Typically, a DNA sample is delivered to the sequencing facility and the data is retrieved through the LMSE web site. The current cost is $6/sample.

Application of High Throughput DNA Sequencing to SAGE analysis

Serial Analysis of Gene Expression (SAGE), is a PCR-based method developed by Bert Vogelstein and Kenneth Kinzler to identify differences in steady-state mRNA levels between two RNA samples. SAGE is based on the idea that the relative proportion of gene-specific expressed sequence tags (ESTs) in a cDNA pool, reflects the relative abundance of the corresponding mRNA transcripts in the original RNA preparation.


In the SAGE strategy, cDNA synthesis is initiated with a biotin-labeled oligo-dT primer which permits physical separation of 3’ ESTs using magnetic beads coated with streptavidin. The two enzymes used for tag delineation are NlaIII which recognizes the sequence CTAG, and BsmFI, a type II restriction enzyme that cleaves 10 bp downstream of the sequence GGGAC. Tag sequencing needs to be performed for EACH sample being analyzed. In most reports, approximately 50,000 tag sequences are analyzed.

SAGE web site for research purposes and data sharing is managed by Johns Hopkins University Oncology Center under the direction of Bert Vogelstein and Ken Kinzler. Zhang et al. (Science 276:1268-72, 1997) is a representative SAGE publication that shows the number of transcripts (Tags) that differ between normal colonic (NC) epithelium and colorectal cancer tumors (TU).

Comparison of expression patterns in CR cancers and normal colon epithelium. A semilogarithmic plot reveals 51 tags that were decreased more than 10-fold in primary CR cancer cells (green), whereas 32 tags were increased more than 10-fold (red); 62,168 and 60,878 tags derived from normal colon epithelium and primary CR cancers, respectively, were used for this analysis.

RNA Expression Profiling using DNA microarrays
The basic idea behind DNA microchips has been to covalently attach known DNA sequences onto a solid support surface in a way that will facilitate rapid hybridization to a fluorescently-labeled pool of DNA (or cDNA) molecules. Since each DNA microchip contains a standardized set of DNA sequences, it is referred to as the probe, whereas, the labeled experimental DNA is called the target. By using laser-scanning and fluorescence detection devices to "read" the chip surface, the hybridization pattern can be quantitatively analyzed to determine the sequence complexity of the DNA target population.

The simplest form of DNA chip technology has been to spot denatured cDNA molecules onto a glass microscope slide using a computer driven robotic arm to precisely position the probe material. The probes consist of known EST sequences that are catalogued by gene identity in a computer database. Pat Brown's lab at Stanford has been one of the leaders in microarray technology.

An excellent example of how DNA microarray technology has been used to identify relevant RNA expression patterns that are associated with disease outcomes, is a recent paper by Alizadeh et al. (Nature 403:503-511, 2000).

The National Human Genome Research Institute (NHGRI) has an extensive multicenter array project focusing on cancer research. Commercial enterprises include Arrayit.com, part of TeleChem International, which is a Silicon Valley start-up company that includes former Stanford postdocs, including Mark Schena.

A second type of DNA microchip has been developed by Affymetrix , a California biotechnology company, using a combination of photolithography and solid phase oligonucleotide chemistry to covalently attach short oligonucleotide probes (25-mer oligos) to a solid support surface. The Affymetrix chips contain 100,000 different oligos in a 4 cm2 area and each probe site has ~10⁷ oligo molecules. Check out this Quicktime movie from Affymetrix.

By designing oligos that span a across an entire exon using a register of 1 nucleotide change between adjacent probe sites, and a window of 25 nucleotides at a time, it is possible to utilize Affymetrix chip technology for DNA re-sequencing. In this re-sequencing strategy, the mid-point nucleotide (number 13 in a 25-mer) is synthesized as an A, G, C, or T.

Hybridization of labeled genomic target DNA to these "SNiP chips" is being used to create human genomic fingerprints for gene mapping studies. Other applications of oligo DNA microchips include the generation of mRNA profile arrays that can be used to measure differences in gene expression levels, similar to what is being done with cDNA microchips.

Does the AMG reference to GATTACA in the flow scheme below ring any bells (hint: Ethan likes Uma).
Check out this Quicktime movie starring the GATTACA invalid ... "there is no gene for the human spirit."

Robotic-driven computerized laboratory workstations
Many of the recent advances in high-throughput genome analysis and gene function bioassays, have included improvements in automated laboratory robots. Examples of lab robots include pipetting devices, colony pickers, microarraying devices and PCR workstations.

By combining robotic pipetting devices, with available computer-controlled robotic arms and automated detection systems, it is now fairly straightforward, albeit costly, to design a fully automated molecular genetic screening assay. Indeed, whole genome sequencing, on the scale of the Human Genome Project, has been dependent on the development of reliable robot-controlled automation.

Beckman Coulter is another instrumentation company with workstation solutions to high throughput.

What factors account for the high throughput capability of automated DNA sequencing using fluorescently labeled dideoxynucleotides?

Design a hypothetical automated robotic workstation that could be used to sequence the genome of the Kartchner Cavern salamander, begining with a shotgun plasmid library plated out on agar plates.

SAGE analysis includes a PCR step to amplify ligated Tags prior to DNA sequencing. Since PCR amplification could skew the perceived frequency of transcript Tags in a sample, it is important to analyze the DNA sequencing data using "nearest neighbor" calculations. Briefly explain how this could be done and what would you be looking for.

In the spirit of the SciFi movie "GATTACA", describe how an Affymetrix type DNA chip might be used in combination with single nucleotide polymorphisms (SNiPs) to create identification profiles of individuals as a genotypic fingerprint.

Assuming enough human SNiPs are mapped for the purpose of genotypic fingerprinting, what would be the limiting factor in designing an instantaneous "finger prick" assay as depicted in the GATTACA movie, and how might this limitation be overcome?