Modified method for double stranded DNA sequencing and synthetic oligonucleotide purification

Abstract

We report here a modified protocol for double stranded DNA sequencing (1) and a simple method for synthetic oligonucleotides purification. Primers were synthesized with a Pharmacia Gene Assembler using cyanoethyl phosphoramidites and modified T7 DNA polymerase (SequenaseTM) was used in sequencing reaction mixtures. Sequencing results as good as single stranded DNA sequencing can be routinely produced ( fig.1). .A Primer preparation: a After synthesis, incubate detritylated primers from the DNA synthesizer with NH40H at 55°C for 16 hours. b Deprotected primer in NH40H is loaded directly onto a Pharmacia Mono Q 5/5 column equilibrated with 10 mM CAPS (3-[Cyclohexylaminoj-1l-propane-OA " -wsulfonic acid) , pH 11, and elute with a NaCI gradient. Set the flow rate and gradient at 0.5 -as ml/min and 10 mM NaCI/min respectively. This al2 gradient can give single base separation for most primers shorter than 30 bases. A chromatogram 2 for the separation of a 18 bases long primer is presented in figure 2. Fig. 2 c. Primer from the column is diluted to desired concentration with water and use directly in annealing step. B Annealing step: a To 2 tg of CsCI-EtBr purified supercoiled DNA, add NaOH to 0.2 M (total volume 10p1), incubate at room temperature for 5 min. b. Add 10 pmoles of primer (7 RI), mix by vortexing. c Neutralize by adding 3 IL of 3 M sodium acetate (pH 4.5), then precipitate the template/primer with 75 g1 of 100% ethanol at -700C for 10 minutes. d Pellet the template/primer by centrifugation, then wash the pellet with 75% ethanol. Recover the pellet by centrifugation at room temperature for 10 min. e Draw away the ethanol, and dry the template/primer in a Sped Vac concentrator. t Resuspend the template/primer in 8 IL H20. -C Sequencing reaction: See SequenaseTM Manual (2) for details and buffer composition. a The reaction mixture consists of: 8 ;1I tamplate/primer in H20, 2 pLI Sequencing buffer, 1 1l 0.1 M DTT, 2 p1 5 fold diluted Labeling Mix, 1 p1 [y-35SJ dATP (20-M, 10 1LCi/p1), and 2 p1t 8 fold diluted SequenaseT. Incubate at room temperature for 5 min after mixing. _= b. Carry out the termination reaction as in (2) at 370C for 5 min.

Identification of mRNA for epidermal growth factor and transforming growth factor-α present in low copy number in human endometrium and decidua using reverse transcriptase-polymerase chain reaction

“…DNA sequencing Double-stranded plasmid DNA sequencing was performed by the dideoxy chain-termination method (Sanger, Nicklen & Coulson, 1977) using a modification of the Sequenase (USB) protocol in which the DNA is denatured at 37°C for 10 min in 0-2 M NaOH, and the primer was annealed to the denatured template before precipitation (Chi, Hsueh, Hui & Tarn, 1988). Following development of the autoradiogram, the DNA sequence was computer aligned with the previously published DNA sequences for EGF and TGFa, using the DNAid program (Dardel & Bensoussan, 1988).…”

Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity

“…Primer extension was performed by annealing 1 pmol of an oligonucleotide (5'-GCCAT`I'lGGACTACCT-3'; complementary to the lac operon mRNA, positions 83 to 99) to 15 ,ug of RNA, followed by cDNA synthesis as previously described (48). Primer-extended products were separated on a 6% polyacrylamide-8 M urea sequencing gel together with the products of a double-stranded sequence reaction (10) obtained with the same primer and pMG820 DNA.…”

Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid

“…After purification on agarose gels, the DNA fragments were inserted into pBAce expression vectors [29] and the resultant plasmids were transformed into E. coli TG1 cells. Fragments encoding the wild-type and mutant GST isoenzymes on the pBAce vector were sequenced in their entirety [30] to ascertain that only the desired mutations had occurred during the manipulations. Expression of GST isoenzymes on pBAce plasmid under the control of phoA promoter has been described in detail [31].…”