Sequenase Version 2.0 DNA Sequencing Kit
Can be used for either internal labeling with α-labeled dNTPs or with 5' end-labeled primers
The Sequenase™ Version 2.0 DNA Sequencing Kit features Sequenase Version 2.0 DNA Polymerase, the standard for high quality manual DNA sequencing(1). Sequenase Version 2.0 DNA polymerase is a genetically engineered form of T7 DNA polymerase which retains extraordinary polymerase activity with virtually no 3'→5' exonuclease activity. It is highly processive, able to effectively incorporate nucleotide analogs for sequencing (dideoxy NTPs, α-thio dATP, dITP, 7-deaza-dGTP, etc.) and is not easily impeded by template secondary structure. The kit includes all the reagents necessary to achieve high quality results (Fig. 1). Use of the specially formulated buffers and mixes included in the kit will maximize yield of sequence information.
The kit can be used for either internal labeling with α-labeled dNTPs (Fig. 2) or with 5' end-labeled primers.
Resolve Gel Compressions with dITP Nucleotide Mixes
The substitution of dITP (deoxyinosine triphosphate) for dGTP in the reaction mix eliminates the secondary structures that produce gel compressions. dITP forms fewer H-bonds with dCTP than does dGTP, so product is more readily denatured during gel electrophoresis. Hence, sequence data is free from gel-based compression artifacts and results are more accurate.
Emphasize Sequence Close to Primers with Mn Buffer
Manganese (Mn) is added to emphasize sequence close to the primer, which may be weak if insufficient DNA template is used. Mn++ increases the incorporation rate of dideoxynucleotides relative to deoxynucleotides. Thus, termination occurs earlier and more sequence is visible close to the primer(2,3).
Increase Read Length with Sequence Extending Mixes
These mixes enable chain terminations to be extended to more than 3,000 bases from the primer. The use of these mixes provides a simple method to further extend the range of sequence, if needed (Fig. 3). Keep in mind that this degree of extension can reach well beyond the limits of any electrophoresis gel resolution, yet use of the mixes when combined with short and long gel runs can increase overall sequence yield.
Eliminate Weak Bands with Pyrophosphatase
Occasionally, weak bands may occur with prolonged reaction times (greater than 5 min)(4,5), or when dITP is used in the sequencing reaction(6). The addition of pyrophosphatase can prevent weak band intensities brought on by sequence-specific pyrophosphorolysis catalyzed by the polymerase.
Convenient Enzyme Storage with Glycerol Enzyme Dilution Buffer
Sequenase DNA polymerase can be pre-diluted to a working concentration for storage of the enzyme in this form. This eliminates the necessity of diluting the polymerase prior to each sequencing reaction. Also, the addition of glycerol enhances the stability of the enzyme in sequencing reactions. Note: Pre-dilution of the polymerase with this buffer results in higher glycerol concentration in the sequencing reaction. A Glycerol Tolerant Gel (GTG) Buffer must be used in the sequencing gel and buffer chambers to eliminate glycerol-induced distortion of bands at approximately 350 to 600 bases beyond the primer(7). If this region is beyond your region of interest, Tris-Borate-EDTA (TBE) Buffer may be used.
Sequenase Version 2.0 DNA Polymerase
Enzyme Dilution Buffer
Glycerol Enzyme Dilution Buffer
Sequenase Reaction Buffer (5X)
Control DNA M13 mp18
Primer (-40 Universal)
Labeling Mix (dGTP, 5X)
ddGTP Termination Mix (for dGTP)
ddATP Termination Mix (for dGTP)
ddTTP Termination Mix (for dGTP)
ddCTP Termination Mix (for dGTP)
Sequencing Extending Mix (for dGTP)
Labeling Mix (dITP, 5X)
ddGTP Termination Mix (for dITP)
ddATP Termination Mix (for dITP)
ddTTP Termination Mix (for dITP)
ddCTP Termination Mix (for dITP)
Sequence Extending Mix (for dITP)
This kit and all the enclosed reagents should be stored frozen at -20°C (NOT in a frost-free freezer). Keep all reagents on ice when removed from storage for use. Sequenase Version 2.0 enzyme must be stored at -20°C. Never store Sequenase enzyme in a frost-free freezer since the temperature rises above 0°C daily. If enzyme dilution buffer (no glycerol) is to be used, only dilute the amount of enzyme which is to be used that day. Dilute into ice-cold buffer and keep on ice until use.
- TABOR, S. AND RICHARDSON C. C. (1989) J. Biol. Chem. 264, 6447-6458.
- FULLER, C. W. (1989) Comments 16, No. 3, United States Biochemical Corp., Cleveland, OH.
- TABOR, S. AND RICHARDSON, C. C. (1989) Proc. Nat. Acad. Sci. USA 86, 4076-4080.
- RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, No. 1, United States Biochemical Corp., Cleveland, OH.
- TABOR, S. AND RICHARDSON C. C. (1990) J. Biol. Chem. 265, 8322-8328.
- TABOR, S. AND RICHARDSON, C. C. (1989) Proc. Nat. Acad. Sci. USA 84, 4767-4771.
- PISA-WILLIAMSON, D. AND FULLER, C. W. (1992) Comments 19, No. 2, United States Biochemical Corp., Cleveland, OH.