Avoiding strand reassociation in direct sequencing of double-stranded PCR products with thermolabile polymerases.

Wanner, R.; Tilmans, I; Mischke, Dietmar

doi:10.1101/gr.1.3.193

Cited by 14 publications

(4 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Direct sequencing of the PCR-ampli¢ed DNA was done in reactions using single-stranded binding protein (US Biochemicals) to prevent strand reassociations (30). The primers used for amplifying exon 1 were: primer 1a(z), 5'-ACT CCT ATC ACT GGT GCA ACC C-3', BK11B(z), 5'-TTG GCT ACA GCT ACG GCG GAG GAT-3' and BK21(^), 5'-TGA GAT CAT CAA TAG TGT TAT AAT-3'.…”

Section: Direct Pcr Ampli¢cation Of K9 Gene and Sequencing Proceduresmentioning

confidence: 99%

Mutations in the 1A Rod Domain Segment of the Keratin 9 Gene in Epidermolytic Palmoplantar Keratoderma

Yang

Lee²,

Kang³

et al. 1998

Acta Dermato-Venereologica

View full text Add to dashboard Cite

Palmoplantar keratodermas (PPK) constitute a heterogeneous group of diseases marked by the thickening of palms and soles of affected individuals. They are divided into autosomal dominant and autosomal recessive groups by the mode of transmission. The autosomal dominantly transmitted group is further divided into epidermolytic (EPPK, Voerner) and non-epidermolytic (NEPPK, Unna-Thost) types according to the histopathologic findings. Recent development of molecular approaches has confirmed that EPPK and NEPPK are caused by the mutations in keratin 9 and 1 genes, respectively. We have studied three families of EPPK to find the mutation in the keratin 9 gene. DNA sequence analyses revealed single base changes in sequences encoding the highly conserved 1A rod domain segment of the keratin 9 gene in two of the three families. These mutations caused Arg (CGG) to Glu (CAG; R162Q) and Arg (CGG) to Try (TGG; R162W) substitutions. The same arginine position has been mutated in the keratin 10 gene in epidermolytic hyperkeratosis, the keratin 14 gene in epidermolysis bullosa simplex, and the keratin 9 gene in hereditary EPPK in Western patients. In this study we show that unrelated Korean patients have similar mutations.

show abstract

Section: Direct Pcr Ampli¢cation Of K9 Gene and Sequencing Proceduresmentioning

confidence: 99%

Mutations in the 1A Rod Domain Segment of the Keratin 9 Gene in Epidermolytic Palmoplantar Keratoderma

Yang

Lee²,

Kang³

et al. 1998

Acta Dermato-Venereologica

View full text Add to dashboard Cite

show abstract

“…Including DMSO (Winship, 1989), formamide (Zhang et aI., 1991), or the nonionic detergents Nonidet-P40 and Tween 20 in Sequenase reactions (Bachmann et aI., 1990), and the latter in Taq polymerase reactions (Innis et aI., 1988) gave good results. Addition of gene 32 protein (gp32, single strand binding protein, or SSBP) isolated from bacteriophage T4 facilitates sequencing with Klenow (Kaspar et aI., 1989) or Sequenase ™ enzymes (Wanner et al, 1992) by binding and stabilizing the single-stranded DNA (available from Pharmacia). Performing the annealing and the sequencing steps in agarose gels appears to inhibit template reannealing, and is possible with Sequenase ™ (Khorana et aI., 1994;Kretz et aI., 1989).…”

Section: Sequencing Linear Double-stranded Dnamentioning

confidence: 99%

Technologies for Detection of DNA Damage and Mutations

Pfeifer¹

1996

View full text Add to dashboard Cite

“…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.…”

mentioning

confidence: 99%

“…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][6][7][8][9][10][11][12][13][14][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.…”

mentioning

confidence: 99%

Direct sequencing of PCR products in agarose ge l slices

Khorana

Gagel²,

Cote³

1994

Nucl Acids Res

View full text Add to dashboard Cite

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

show abstract

Avoiding strand reassociation in direct sequencing of double-stranded PCR products with thermolabile polymerases.

Cited by 14 publications

References 5 publications

Mutations in the 1A Rod Domain Segment of the Keratin 9 Gene in Epidermolytic Palmoplantar Keratoderma

Mutations in the 1A Rod Domain Segment of the Keratin 9 Gene in Epidermolytic Palmoplantar Keratoderma

Technologies for Detection of DNA Damage and Mutations

Direct sequencing of PCR products in agarose ge l slices

Contact Info

Product

Resources

About