Sequenase

Sequenase Parameter Optimizations. The tabulation below provides a summary of Sequenase parameter optimizations.46... 

S pM (corresponding to 1 3 dilution of the Sequenase kit) gave better results than the standard reaction mixture for greater amounts of template, higher dNTP concentrations were required (up to the standard 7.S pM concentration) 15 min at 37°... 

Add 5 pi stop solution (Sequenase kit). Heat for 3 min at 90° before loading sequencing gels. 

Fig. 2. DNA sequences across a homopolymer stretch in the control region of the mitochondrial genomes from two salmon. (A) Ten T residues in pink salmon (Oncorhynchus gorbuscha) and (B) 14 T residues in rainbow trout (Oncorhynchus mykiss). Lanes for each species are from left to right G, A, T, and C. Sequencing templates were prepared by asymmetric amplification directly from purified mitochondrial DNA. Sequencing reactions were performed with Sequenase and the products separated in 6% polyacrylamide, 7 M urea gels.

Sequenase Version 2.0 Step-by-step Protocols for DNA Sequencing with Sequenase Version 2.0, 5th Ed. United States Biochemical, Cleveland, Ohio, 1990. 

There are also a number of new dye-modified analogues for use in FRET analysis. A new four colour set of FRET dideoxy 5 -triphosphate terminators has been developed involving a rigid, linear ET cassette linker chemistry, and formulated with Thermo Sequenase II DNA polymerase. An alternative approach for a four colour set of FRET dyes has been reported where the dyes are incorporated into ODNs as phosphoramidite derivatives. ... 

Fig. 7.18. End-labeling can be achieved by different approaches. Kinasing (I) is most effective with 5 -OH overhangs. In the presence of excess ADP, radioactive phosphate can also be exchanged with a 5 phosphate. Whereas kinasing introduces at most one label per DNA end, tailing (II) allows multiple labels to be introduced (Co is necessary with recessed ends). Fill-in of restriction sites (III) also yields 3 -end labeling. RTase or sequenase should then be used. Oligomers are required if the 5 -end is recessed (IV).

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

Sequenase II kit (US Biochemical Corp., Cleveland, OH) and sequencing apparatus. 

Sequence the insert to ensure that no PCR errors occurred using Sequenase II or a similar product according to the manufacturer s instructions. 

Sequenase. [U.S. Biochemical] T7 DNA polymerase enzyme for dideoxy DNA sequencing. 

IX Thermo Sequenase Concentrated Reaction Buffer (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, cat. no. E79000Y) or equivalent. Store at -20°C. 

IX Thermo Sequenase Concentrated Reaction Buffer 250 pM of each ddNTP... 

---