The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction

A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.     

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.     

Direct sequencing of double-stranded polymerase chain reaction-amplified 16S rDNA.

A number of different procedures have been developed for direct sequence analysis of PCR products. These methods rely on the cumbersome isolation of specific PCR products from agarose gels or the production of single-stranded template DNAs. In the approach presented here, we describe primers for the amplification of 16-S rDNA and a simple preparation of PCR product for sequencing.     

DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction.

In its basic concept, in vitro DNA amplification by the polymerase chain reaction (PCR) is restricted to those instances in which segments of known sequence flank the fragment to be amplified. Recently, techniques have been developed for amplification of unknown DNA sequences. These techniques, however, are dependent on the presence of suitable restriction endonuclease sites. Here, we describe a strategy for PCR amplification of DNA that lies outside the boundaries of known sequence. It is based on the use of one specific primer, homologous to the known sequence, and one semi-random primer. Restriction sites in the 5' proximal regions of both primers allow for cloning of the amplified DNA in a suitable sequencing vector or any other vector. It was shown by sequence analysis that the cloned DNA fragments represent contiguous DNA fragments that are flanked at one side by the sequence of the specific primer. When omitting the semi-random primer, a single clone was obtained, which originated from PCR amplification of target DNA by the specific primer in both directions.     

Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing

A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.     

Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction

A method to rapidly generate single stranded DNA for dideoxy sequencing following the polymerase chain reaction is described. By incorporating biotin in one of the amplification primers, we are able to physically separate the two DNA strands produced in the polymerase chain reaction. After amplification, the mixture is passed through a column containing streptavidin agarose. The strand produced by the biotinylated primer is bound in this matrix. The unbiotinylated strand is eluted with 0.2 N NaOH and sequenced by the dideoxy method. This method was utilized to sequence mitochondrial DNA from crude genomic DNA and to determine the sequences of four clones containing human mitochondrial DNA as a test of its accuracy. The use of biotin-facilitated separation permitted us to amplify and sequence DNA samples in a single day.     

DNA diagnosis of hydatidiform mole using the polymerase chain reaction

Summary We have used the polymerase chain reaction (PCR) technique for the diagnosis of hydatidiform mole, a trophoblastic disease. For this, we targeted the hypervariable 3 flanking region of the APOB gene (APOB/ VNTR) because of its high heterozygosity index (0.61) in the Japanese population. We examined seven clinical cases which were tentatively diagnosed as hydatidiform moles. Five of these revealed DNA segments unique to the paternal APOB allele, allowing us to diagnose a complete mole. The PCR technique for targeting the APOB/VNTR appears useful for early diagnosis of hydatidiform mole.     

Human DNA quantitation using Alu element-based polymerase chain reaction

Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng. Multicopy Alu elements include recently integrated subfamilies that are present in the human genome but are largely absent from nonhuman primates. Here, we present two Alu element-based alternative methods for the rapid identification and quantitation of human DNA, inter-Alu PCR and intra-Alu PCR. Using SYBR green-based detection, the effective minimum threshold level for human DNA quantitation was 0.01ng using inter-Alu- and 0.001ng using intra-Alu-based PCR. Background cross-amplification with nonhuman DNA templates was detected at low levels using inter-Alu-based PCR, but was negligible using intra-Alu-based PCR. These Alu-based methods have several advantages over currently available systems. First, the assays are PCR based and no additional unique equipment is required. Second, the high copy number of subfamily-specific Alu repeats in the human genome makes these assays human specific within a very sensitive linear range. The introduction of these assays to forensic laboratories will undoubtedly increase the sensitivity and specificity of human DNA detection and quantitation from complex sources.     

Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.

The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.     

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.     

Hot start of the polymerase chain reaction using DNA helicase]

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.     

Direct sequencing of long polymerase chain reaction fragments

Direct sequencing of polymerase chain reaction (PCR)-generated templates is a commonly used technique in molecular biology laboratories. We describe an improved method for direct sequencing of PCR fragments longer than 20 kb obtained with a commercial mixture ofTaq andPwo DNA polymerases. The sequencing protocol was optimized for an automated infrared DNA sequencer, consistently yielding long reads (500–600 bases).