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DNA Sequencing

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DNA Sequencing: The DNA sequencing service accepts plasmid, single-stranded DNA, BAC DNAs, or PCR products as templates. Sequencing reactions are performed with any primer provided by the user. Reactions are performed by a cycle sequencing protocol with AmpliTaq™ DNA Polymerase using ABI BigDye™ Terminators. Modified protocols and kits are used for difficult or very large templates. Sequencing data, both raw data in a data in a waveform format and called nucleotide sequence, are immediately sent to researchers by email. Color-printed waveforms are provided by request. Data from completed runs are securely archived on compact discs for future access by users.

Automated fluorescent capillary sequencing provides much longer reads (generally 400 to 850 nucleotides) from a single sample than can be achieved by manual sequencing methods. Results are generated quicker and more accurately than slab gel electrophoresis platforms. Additionally, electronic transcription of data eliminates errors inherent in manual transcription.

To request this service, the investigator provides the mixed DNA sample and primer with a completed request form. An alternate option for the researcher is to perform their own sequencing PCR reactions using the fluorescent dye kit sold at cost by the Facility to users. These “Premade” samples are sent to the Core Facility, analyzed on the Capillary Sequencer, and results emailed to the end user. This results in 250% cost-savings to users.

DNA Fragment Analysis: The DNA Sequencing Core Facility has the Genescan and Genotyper software packages necessary for microsatellite analysis such as LOH and MIN. The Facility’s staff is highly experienced in running these assays, freeing the user to focus on the application of these technologies in their research. The SNP analysis utilizes Applied Biosystems’ SNaPshot primer extension kit. With this software and reagents, the ABI 3100 can analyze 160 multiplexed loci in 30 minutes.

Real-Time PCR:
The Core Facility operates an ABI 7900HT Real-Time PCR system. This system can accommodate any and all real-time PCR applications. The 7900HT is a high-throughput system which contains robotics enabling multiple 384 or 96 well plates to be loaded automatically. Continuous wavelength detection from 500-660 nm allows the use of multiple fluorophores in a single reaction. The software is designed to perform all necessary calculations including dissociation curve analysis, absolute quantitation, and relative quantitation using standard curve or comparative CT algorithms.

Possible applications include:

  • Allelic Discrimination:SNP Analysis using fluorescent 5' nuclease assay
  • Measurement of gene expression levels (gene expression quantitation)
  • Quantitative PCR
  • Array Verification
  • Gene Expression
  • Analysis of mRNA Expression
  • Mutation Detection
  • Detection and quantitation of nucleic acid sequences

Using the Applied Biosystems TaqMan® Genotyping Assays, large-scale screening of known SNPs can be accomplished in a very short amount of time. These assays contain two TaqMan® probes, one for each allele, multiplexed into one reaction well. The 7900HT software rapidly detects end-point fluorescence for each allele and displays the data in tabular and plot formats.

Oligonucleotide Synthesis: The DNA Sequencing Core Facility has an outsourcing arrangement with Integrated DNA Technologies (IDT) of Coralville, IA. This company provides standard oligonucleotides with a guaranteed two-day turn-around time and can synthesize over 9,000 oligonucleotides per day. Furthermore, at least one and often two quality control checks are performed on all oligonucleotides. Standard DNA oligonucleotides are discounted up to 33%. Modifications, purifications, RNA synthesis and Dual-labeled probes are available at a 10% discount off of list price. Additionally, the $12 shipping charge individual laboratories would pay per order is waived.

To use this service, the requester submits an oligonucleotide-synthesis request form electronically via the DNA Shared Resource website. The web-based ordering system is a private site developed for the Facility by IDT and is directly linked to the facilities at IDT, such that all submissions go directly to the synthesizers to eliminate errors during handling. Users are notified when oligos arrive by email. Most users prefer to pick up oligonucleotides in person as soon as they are ready. Upon request, orders can either be shipped by campus mail or hand delivered to New Brunswick via the RWJMS run - same day courier service.

ABI Bulk Freezer Program: The DNA Core Facility is able to purchase select Applied Biosystems reagents and consumables in bulk and pass this significant cost-savings onto users. Items are stocked at the Core Facility and are available for pick-up or can be delivered to New Brunswick via the RWJMS courier service. This saves researchers a shipping fee as well as valuable time.