|Date: November 10, 1997||Chart 1|
The kits profiled exploit two methods for labeling sequencing products: the incorporation of fluorescent dye-labeled primers and the incorporation of dye-labeled dideoxynucleotides (ddNTPs), or dye terminators. The use of dye-labeled primers is the most common method because the modified forms of Taq and the other novel polymerases used in these kits discriminate against dye-labeled ddNTPs and are unable to efficiently incorporate them into the reaction products. Expressly engineered polymerases, on the other hand, such as ABI's AmpliTaq DNA Polymerase, FS and Amersham's Thermo Sequenase, have been developed to incorporate dye-labeled ddNTP's more efficiently. These engineered enzymes have a decreased discrimination function against ddNTP incorporation that not only makes the use of dye-terminators possible, but also improves the dye-primer chemistry as well, resulting in even signal intensities for both.
Opinions differ about which methodology is best. Fluorescent-labeled ddNTP procedures are probably the least expensive, especially if several primers are needed to sequence a particular template, but this use requires knowledge of the downstream sequence to design those primers. In contrast, the protocols for fluorescent-labeled primers are reportedly faster and touted to produce more accurate and reproducible results. However, these methods are frequently limited by the range of available fluorescent primers and the expense required to synthesize them.
Most of the kits belong to the cycle sequencing category and the remainder use classical, isothermic incubations. A few, such as the SequiTherm kits from Epicentre Technologies and the IR Taq DNA Sequencing Kit from Boehringer Mannheim, function well in both modes. Regardless of the mode, however, the number of bases read past the primer under optimal conditions, as reported by some manufacturers, now exceeds 1,000. PE Applied Biosystems reports a survey stating that te average read length in the United States is about 500 bases, which is probably a better indication of the actual read lengths produced considering the various labs and skill levels involved.
While the usual purpose of these kits is to sequence general DNA templates, kits with specific applications are creeping into the marketplace. Visible Genetics, Inc., has recently introduced kits for sequencing specific Human Leukocyte Antigen (HLA) genes important for tissue typing in bone marrow transplants, as has Pharmacia. Visible Genetics also has several other kits with specific applications under development, including HIV genotyping.
Shown below are brief profiles of the eight companies and their automated sequencing kits.
AmershamTM Life Science
Perhaps the most frequently recommended for fluorescent automated sequencing applications, Amersham's kits are designed around Thermo SequenaseTM, a heat-stable exonuclease-free DNA polymerase. Thermo Sequenase possesses a genetic modification that removes discrimination against ddNTP incorporation, resulting in even signal intensities for fluorescent cycle sequencing applications. Additionally, Amersham has developed the unique DYEnamic Energy Transfer (ET) primers. In these primers, two fluorescent dyes are attached to each primer molecule, reportedly creating much stronger signal strengths than single-dye primers. The stronger signals confer longer read lengths, reduced cycle times, and reduced template requirements to reactions using ET primers.
Amersham sells two groups of kits. The kits of the first group, known collectively as the DYEnamic Direct Dye-Primer Cycle Sequencing Kits, contain either dGTP or 7-deaza-dGTP*, and include premixes for A, C, G, and T reactions (containing Thermo Sequenase, reaction buffer, dNTPs, and ddNTPs), control pUC18 DNA, and a new bright pink formamide loading dye. Some kit variants are premixed with either -21 forward or -28 reverse ET primers. All are optimized for Thermo Sequenase and ET primers and provide high signal intensities. In many applications, users may directly load their sequencing reactions onto a gel without performing any ethanol precipitations.
Amersham's Thermo Sequenase Dye-Terminator Cycle Sequencing Kits comprise the second group and consist of a premix and a core kit. The reagent in the premix kit contains Thermo Sequenase, reaction buffer, dNTPs, and dye-labeled ddNTPs, while the core contains each of these reagents separately. Both kits contain control M13mp18 DNA, universal primer, ammonium acetate, and the new bright pink formamide loading dye, and both reportedly provide high-quality sequence data and ease of use.
*7-deaza-dGTP is licensed from Boehringer Mannheim GmbH All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Group that supplies them. A copy of these terms and conditions is available upon request.
Amersham, Thermo Sequenase, Nucleix Plus, Redivue, and DYEnamic are trademarks of Amersham International plc.
Boehringer Mannheim produces the IR Taq DNA Sequencing Kit for standard and cycle sequencing of single- and double- stranded DNA templates such as PCR products, lDNA, and cosmid DNA. Incorporating infrared-labeled primers, Taq polymerase, and 7-deaza-dGTP, this kit had been designed expressly for the LI-COR 4000S and 4000L automated DNA sequencers.
The Long Range Termination Set for Taq Sequencing consists of four long-range termination mixtures designed to generate extended sequence ladders up to 1,000 bases in length. The mixtures function in combination with standard and cycle sequencing reactions of single- and double-stranded DNA templates.
Boehringer Mannheim also provides an extensive product range of primers, nucleotides, and sequencing enzymes for specific, customized sequencing applications.
The SequiTherm EXCEL II Long-Read DNA sequencing kits from Epicentre are built around common benefits. All three kits use up to five times less template than earlier versions; perform both cycle and classical (isothermal) sequencing reactions; read through difficult templates such as regions of high GC content, hairpin loops, and dinucleotide repeats; and produce up to 1,200 bases of sequence data per run. As their names imply, all three include Epicentre's SequiTherm EXCEL II DNA Polymerase, a sequencing enzyme designed to improve signal intensities.
The SequiTherm EXCEL II Long-Read DNA Sequencing Kit-LC and the SequiTherm EXCEL II Long-Read Premix DNA Sequencing Kit- LC have been optimized for use with the LI-COR 4000 and 4200 IR2 automated DNA sequencers using fluorescent-labeled primers. The recently introduced Premix format combines the buffers, termination mixes, and SequiTherm EXCEL II DNA polymerase so that only the primer and template need to be added during set up. Both kits offer the choice of performing single primer or differentially labeled two-primer reactions. The two-primer reactions simultaneously produce more than 1,000 bases of data from both directions.
The SequiTherm EXCEL II Long-Read Premix DNA Sequencing Kit- ALF has been optimized for the ALF automated DNA sequencers from Pharmacia. The Premix format combines reagents to simplify reaction assembly.
All three kits include enough reagents for 100 reactions.
Operating from the United Kingdom, GENPAK Limited manufactures three fluorescent DNA sequencing kits and related products. The FLASH, LASH, and AASH kits provide all the reagents necessary for fluorescence-based dye primer sequencing reactions using single- or double-stranded templates for the ABI automated sequencers, the LI-COR automated sequencers, and the ALFexpress automated sequencing system, respectively. These three kits all include enzyme, premixed A, C, G, and T sequencing cocktails containing a stabilizer, and loading buffer.
For fluorescent dye terminator sequencing, GENPAK Limited sells a unique, specially formulated sequencing reagent called halfTERM. The addition of an equal volume of halfTERM to sequencing reactions using ABI or Amersham dye terminators results in at least a 40% reduction in dye terminator costs. According to GENPAK, the read length, sequence accuracy, and resolution obtained with halfTERM are the same as those obtained from unmodified reactions. Also available from GENPAK is the halfTERM XL Dye Terminator Sequencing Reagent, which was formulated to provide robust sequencing of BACs, P1s, and cosmids. Free samples of terminator reagents are available through GENPAK Limited's e-mail address at email@example.com.
PE Applied Biosystems
PE Applied Biosystems has recently announced the availability of a new system of fluorescent dyes called BigDyes. BigDyes are single energy transfer molecules consisting of an energy donor and acceptor dye connected by a highly efficient energy transfer linker. The acceptors are four new dichlororhodamine dyes (ABI Prism dRhodamines) which are improvements over the conventional rhodamine dyes. Because dichlororhodamine dyes exhibit less spectral overlap at their maximum excitation efficiencies, they produce less background noise, cleaner signals, and improved base calling accuracy at longer read lengths than the conventional dyes.
The BigDyes have been incorporated into both dye-terminator and dye-primer kits that also feature AmpliTaq DNA Polymerase, FS. These new kits are suitable for most sequencing applications. Coupled together, BigDyes and AmpliTaq DNA Polymerase, FS represent an exciting advancement in fluorescent automated sequencing kit technology.
The dRhodamine Terminator Cycle Sequencing Kit features terminators labeled with the four new dRhodamine dyes in place of PE Applied Biosystems' conventional rhodamine dyes. The result is a kit that produces a much higher signal-to- noise ratio, improved base calling, and longer read lengths.
PE Applied Biosystems' Dye Primer and Dye Terminator kits feature primers and terminators labeled with its conventional rhodamine dyes.
The terminator kits are supplied in a ready reaction format consisting of AmpliTaq DNA Polymerase, FS, dNTP's, dye ddNTP's, and buffer pre-mixed into one tube. The primer kits include four pre-mixes (A,C,G,T). Each pre-mix contains AmpliTaq DNA Polymerase, FS, dNTP's, 7-deaza-GTP, ddNTP's, and dye primers. Kits are available with reagents for 24, 100, 1000, or 5000 sequencing reactions.
For routine sequencing of double-stranded plasmid DNA templates on the ALF/ALFexpress DNA sequencers, Pharmacia sells the AutoRead and Cy5 AutoRead Sequencing kits. Using T7 DNA polymerase and optimized extension buffers, the AutoRead kits employ a simple, one-step method and fluorescent primers to sequence multiple templates in a microtiter plate.
Pharmacia has designed the AutoCycle and Cy5 AutoCycle Sequencing Kits for cycle sequencing of amplified DNA and cosmid templates on the ALF/ALFexpress DNA sequencers. Templates are sequenced with Taq polymerase and fluorescent primers using a simple and effective protocol.
All of the AutoRead and AutoCycle kits contain 7-deaza-dGTP* to minimize band compressions, two fluorescent-labeled primers for direct sequencing of M13/pUC-based templates, and reagents sufficient for 100 reactions. For large-volume users, 200- and 1000-reaction kits are available, as well as materials to synthesize custom fluorescent primers.
Pharmacia's AutoLoad Solid Phase Sequencing Kit has been designed for direct sequencing of PCR products up to 800 base pairs in length on the ALF DNA sequencers. PCR products amplified with a biotinylated primer are captured on a specially designed, streptavidin-coated sequencing comb and serve as the sequencing template for reactions using dye- labeled primers. The resulting sequencing products remain bound to the comb until it is transferred to the sequencing gel. This kit includes reagents for 100 reactions and 7-deaza-dGTP.
Finally, Pharmacia has recently introduced the HLA DRB Typing Kit for automated HLA subtype determination of the DRB1 and DRB3/4/5 genes using the ALFexpress or ALF DNA sequencers.
*Sold pursuant to a license from Boehringer Mannheim GmbH. 7-deaza-dGTP is covered by U.S. Patent No. 4,804,748 issued to Boehringer Mannheim GmbH.
Takara Shuzo Co., Ltd.
Distributed by PanVera Corporation of Madison, Wis., the Ladderman Dideoxy Sequencing Core Kit is a product of Takara Shuzo Co., Ltd., of Japan. Designed around Bca DNA polymerase from the thermophilic bacteria Bacillus caldotonax, the Ladderman Kit is intended for use with the automated sequencers from Pharmacia, ABI, and LI-COR. The kits use fluorescent-labeled primers to sequence M13-derived single- stranded phage or phagemid DNA and double-stranded plasmid DNA templates. The reactions consist of an isothermic incubation at 65¡C for 15 minutes, followed by an ethanol precipitation step. Alternatively, a single-cycle thermal cycler procedure is included for primers whose TM values or structures are not suitable for reactions at 65°C.
On the average, the Ladderman Dideoxy Sequencing Core Kit produces 940 bases of data when used with LI-COR instrumentation. The kit consists of enough materials for 100 four-lane reactions, although the fluorescent primers are not included.
Visible Genetics, Inc.
Visible Genetics, Inc. (VGI) develops, manufactures, and markets automated DNA sequencing systems designed to analyze genes linked to disease. The company offers fully integrated sequencing kits based on PCR and optimized for clinical DNA sequencing and analysis using its OpenGeneTM automated sequencing system.
Recently, VGI announced the availability of four new DNA GeneKits designed for sequenced-based research of the A, B, and C loci of the Class I, and the DRB1 locus of the Class II, HLA genes. These genes are important genetic markers used for matching bone marrow transplant donors and recipients. These kits represent an important advance in ensuring proper matches.
VGI's TruGene HIV Genotyping GeneKit is a fluorescent primer- based kit currently being tested. This kit is designed to sequence the Protease and RT regions of the HIV genome using CLIPTM, a DNA sequencing technique designed to directly sequence small amounts of amplified templates. The highly variable RT and Protease regions are important to the virus's ability to develop drug resistance. Other kits in development include tests for RB1, a gene associated with eye cancers in children; p53, a gene found in solid tumors in many cancers; and the human papilloma virus, which is believed to be responsible for cervical cancer.
Pushed onward by the continuing demands for faster and longer sequence reads, sequencing kit and instrumentation manufacturers constantly upgrade their products. Indeed, several of the marketing and product managers interviewed during the preparation of this profile mentioned that their companies are working hard at developing new polymerases and other ideas to improve automated DNA sequencing. Particularly noteworthy is the introduction of sequencing kits with specific clinical and other applications. So be on the lookout for new products as fluorescent automated DNA sequencing technology continues to improve and expand into new territories.
Michael Brush is an Associate Scientist and Radiation Safety Officer at Hitachi Chemical Company in Irvine, Calif. He can be reached online at MDBrush@compuserve.com.