Direct sequencing of PCR products in agarose ge l slices

Abstract

Direct DNA sequencing of amplified polymerase chain reaction (PCR) products offers several advantages over cloning of amplified DNA products. It is faster (1 day versus 3-5 days) and in DNA samples containing sequence polymorphisms both the normal and mutated sequence can be detected in the same sequencing reaction. The major problems encountered in direct sequencing of amplified DNA have been contamination by PCR primers and deoxynucleotides, and overcoming fast template renaturation. Techniques which have been used to purify the amplified product include: differential precipitation, electrophoretic purification, absorption of DNA to specific matrices, or differential filtration (i.e. centricon) (1-6). Each of these techniques is capable of improving the quality of sequence data, but all are time consuming and subject to some perfonmance variability. The problem of template reannealing has also been approached by several methods including: rapid cooling following denaturation, use of thermal stable polymerases, increased primer concentration and preparation of a single-stranded DNA templates by: asymmetric PCR, biotinylation of one primer or use of strandspecific nuclease (2,5,7,8). We have devised a strategy for direct sequencing of PCR products using a simple modification of the standard Sequenase@ protocol (U.S. Biochemicals, Cleveland, OH).The major change in the protocol is that template denaturation, primer annealing and DNA sequencing are all performed in the presence of agarose. This technique simplifies purification, eliminates the necessity for elution of the DNA, and may act to prevent reannealing of the DNA template. The method described here was developed to allow rapid analysis of DNA samples from patients with multiple endocrine neoplasia type 2 (9). These patients have heterozygous mutations in one of six codons in the RET proto-oncogene (10,11). DNA sequence analysis permits rapid determination of specific mutations contained in one of three exons. PCR products (2-3 ,tg) were purified by electrophoresis on 0.8-1 % SeaPlaque® (FMC, Rockland, ME) agarose gels.Electrophoresis was performed with either TrisAcetate or TrisBorate buffer, in the absence or presence of ethidium bromide. We used an Owl Scientific (model B1A) apparatus with a 6 well 1.5 mm comb. Following electrophoresis the DNA band of interest was excised, taking care to remove as much excess agarose as possible. The gel slice containing the PCR product was weighed, an equal volume of dH2O was added, and melted at 75°C for 5-15 minutes. The annealing reaction was prepared by combining: 5-10 Al of melted gel containing template DNA, 1 jd of sequencing primer (10 A260/ml), and dH2O to 11 (4. This mixture was denatured by incubation at 100°C for 3 minutes and annealing was performed by immediately placing the tube in ice for 5 minutes. Dideoxy-DNA sequencing was performed on 10

Phase I Study of the Farnesyltransferase Inhibitor BMS-214662 Given Weekly in Patients with Solid Tumors

“…In the first patient, with a germ line codon 634 RET proto-oncogene mutation (26), there was a reduction in serum CT from 479,000 to 208,000 pg/mL with a return to a value of 401,000 pg/mL following discontinuation of therapy. In the second, a patient with a codon 918 germ line RET mutation (27), there was a reduction in the serum calctionin from 68,000 to 15,000 pg/mL during therapy with a value of 71,000 pg/mL following discontinuation of therapy and accompanied by a reduction in her baseline bone pain, flushing episodes, and diarrhea.…”

Mutations in Genes Encoding the Mitochondrial Outer Membrane Proteins Tom70 and Mdm10 of Podospora anserina Modify the Spectrum of Mitochondrial DNA Rearrangements Associated with Cellular Death

“…The sequence of the mutant allele of the gene was determined after PCR amplification of various fragments overlapping the gene. The PCR fragments were directly sequenced according to the method of Khorana et al (19). The presence of the mutation was ascertained by sequencing two independent PCR fragments on both strands.…”

Pseudoachondroplasia due to the substitution of the highly conserved Asp482 by Gly in the seventh calmodulin-like repeat of cartilage oligomeric matrix protein

“…Dideoxy-DNA sequencing reactions were undertaken on the PCR products in agarose gel slices (Khorana et al, 1994). Both DNA strands were sequenced using primers 1-4 as sequencing primers.…”