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As a standard molecular biology technique, PCR uses DNA polymerase to detect, amplify and manipulate DNA targets. Due to its effect of exponential amplification, PCR can achieve high sensitivity required for detecting targets of low abundance. Therefore, it has become the method of choice for the majority of nucleic acid-based tests. In PCR reactions, DNA templates are first unwound into single strands, followed by a quick temperature drop when transient intramolecular secondary structures may form first within the single-stranded templates due to reaction kinetics. In this study, we showed that the adverse effects of stem-loop structures on PCR performance were directly correlated with their thermal stability. Moreover, fractions of intermediate PCR products of templates with stable stem-loop structures were significantly shorter than those without. It was further demonstrated that when encountering the duplex region of such a structure during the PCR extension step, the endonuclease activity of Taq DNA polymerase mediated by its 5'-3' exonuclease activity could digest template strand, resulting in stem-loop structure unwinding and subsequent completion of replication to produce truncated products. This work thus provided some new mechanistic insights into the complex nature of PCR assays, a frequently encountered but neglected aspect of this widely used technique.

Classical hereditary hemochromatosis involves the HFE -gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G > A; rs1800562) and HFE p.H63D (c.187C > G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE -p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE -SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G > A, Coagulation factor V-gene F5 p.R506Q (c.1517G > A; rs121917732), Mitochondria SNP: mt7028 G > A, Mitochondria SNP: mt12308 A > G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G > T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A > G; rs1695), LXR g.-171 A > G, ZNF202 g.-118 G > T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility.

Technical difficulties in full-length cDNA cloning hinder successful characterization of many unknown and potentially novel expressed sequence tags (ESTs). We presently describe improved methods for cDNA cloning. This scheme is based on the polymerase chain reaction and utilizes a degenerate stem-loop annealing primer (dSLAP), consisting of a stem-loop structure followed by 12 random nucleotides, and is called the C-ORF (complete open reading frame) technique. The dSLAP is designed to anneal to first-strand cDNA, while suppressing second-strand synthesis from internal sites because of its bulky stem-loop structure. The C-ORF technique consists of three steps: reverse transcription, dSLAP annealing plus the second-strand synthesis, and PCR amplification. Applications of dSLAP to both known and previously unknown cDNA targets resulted in cloning of their complete open reading frames, in most cases after a single application of the C-ORF method. The currently described protocol is simple and does not require unusual molecular biology reagents, except for reverse transcriptase, Taq polymerase and a DNA primer, which makes it readily amenable for cloning purposes in individual laboratories. Moreover, this approach has wide applicability and in principle can be used to identify the protein-coding region of virtually any gene in which limited or incomplete sequence information is available.

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF.

KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus (Taq) DNA polymerase I. As expressed from a gene construct in Escherichia coli, translation initiates at Met236, bypassing the 5'----3' exonuclease domain of the DNA polymerase-encoding gene. A sensitive forward mutation assay was used to measure the relative number of mutations introduced into the entire lacZ gene by the polymerase chain reaction (PCR) under various conditions which allow the amplification of such a large DNA span. Two selectable markers, one at each end of the test lacZ fragment, were employed to avoid the plating and scoring of PCR artefacts such as primer initiation in the midst of the lacZ gene, and cloning artefacts such as empty vector plasmid. The measured relative mutation rate was twofold lower for KlenTaq as compared to the full-length Taq DNA polymerase.

Polymerase chain reaction (PCR) was performed with two polymerases, Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenasetm). Both polymerases were used to amplify the same portion of the human 18SrRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenasetm did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (−)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures. Recombinant DNA; hairpin; ribosomal RNA

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.

Nineteen olfactory receptor (OR) genes were isolated from three OR subfamilies in two species of marmoset (Callithrix). Olfactory receptor 912-93 has high sequence similarity among marmosets and between marmosets and humans, suggesting strong conservation of function. All of the remaining seventeen OR genes identified from subfamilies 3A and 1E were pseudogenes. Following pseudogene formation, marmoset OR genes in both 1E and 3A subfamilies underwent duplications, indel events and a high rate of nucleotide substitution. These results provide a contrast to previous studies, and show that in spite of the keen olfactory sense of marmosets, they harbour many OR pseudogenes. A high rate of in vitro recombination using Pfu polymerase but not Taq polymerase was confirmed. The rapid molecular evolution of OR pseudogenes suggests that they do not provide a useful source of sequence variation for conversion to intact OR genes over evolutionary timescales. The overall pattern of OR evolution in marmosets is comparable to the 'birth-and-death' model of gene family evolution. An unbiased view on the evolutionary timing of the reduction of the functional olfactory repertoire in humans must await more data.

Amplification of portions of the intergenic spacer between the katE gene and cryptic cel operon of Escherichia coli was accomplished by the polymerase chain reaction using the DNA polymerase from Thermus aquaticus. Nine different segments were amplified and cloned without error, but one 83-bp fragment was amplified with a high error rate such that 32 of 34 selected clones had three or more nucleotide changes from the expected sequence. The changes were all located in two 9-bp segments immediately adjacent to the 3'-ends of the two primers. Moving the end points of the primers to increase the spacing between them resulted in the isolation of significantly fewer error-containing products. It is proposed that stem-loop structures in the template immediately downstream from the primers interfere with an early stage of elongation and cause misincorporation. This is supported by the observation that destabilisation of one of the stem-loop structures reduced the frequency of errors.

The location, structure and nature of the cos site of the Lactococcus lactis bacteriophage sk1 was determined using a Taq DNA polymerase runoff sequencing technique. The cos site contains a single-stranded 3′ overhang of 11 nucleotides. The region surrounding cos contains several features which may be involved in the binding and catalytic action of a phage terminase. These include four putative terminase-binding sites which show some homology to λ R-sites, an 11-bp direct repeat, a 10-bp inverted repeat, a string of eight consecutive C residues and six copies of the pentanucleotide, AATCT. The spacing between adjacent copies of the pentanucleotides would place them on the same side of the DNA helix.

We have cloned by the polymerase chain reaction a 2.1-kb fragment carrying heavy-chain joining (JH) gene segments and a part of the JH-Cμ intron of the rat. Sequencing showed that the rat genome has four functional JH segments and that only a slight divergence has occurred after the separation of rat and mouse. A systematic sequence comparison between the two species and human revealed an additional JH pseudogene in rat and mouse 5′ of JH1 which has not been described so far. The implications in evolutionary terms are discussed. In addition, we give an assessment of the misincorporation rate of the Taq polymerase.

We report the ability of Taq polymerase to directly transcribe RNA templates in vitro. We have made use of this finding to develop a single-step protocol for amplification of RNA transcripts. The method was shown to require only subnanogram amounts of total cellular RNA as starting material. A microassay was developed in which RNA can be extracted from one drop of blood or 1000 cultured cells, and analyzed for the expression of a specific gene.

A method is described for the direct cloning of DNA fragments amplified by the polymerase chain reaction (PCR). An oligodeoxyribonucleotide, bearing two engineered XcmI sites placed in tandem, was used to generate cloning vectors bearing single 3' deoxythymidine (dT) overhangs at their ends. These 3' dT overhangs are compatible with the 3' deoxyadenosine overhangs found on most Taq polymerase-amplified PCR products. Consequently, Taq polymerase-amplified PCR products can be ligated directly into these modified restriction sites.

More than half of the products of PCR contain an extra A residue at the 3' end, which is the result of the template-independent activity of Taq polymerase. To facilitate cloning of the products of PCR without modification, T vectors, which have a single overhanging T residue at the 3' end, have been developed. In the present study, we constructed new T vectors which can be prepared in the laboratory by simple digestion with the restriction enzymes AspEI or Eam1 105I.

We have devised a strategy to extend the use of the polymerase chain reaction (PCR) to amplify double-stranded DNA when sequence information is available only at one extremity. The only information required is a short stretch of sequence used to design a gene-specific primer, which is then used in combination with a second generic vector primer at the unknown end. The primers are used in a PCR reaction after ligating the unknown end to a generic vector. Restriction, ligation, amplification and sequencing of the products can be achieved within three days. This method eliminates the laborious steps of shotgun cloning, colony screening and culturing of cells. We have used this method to take two contiguous steps beyond the histidine transport operon in Salmonella typhimurium. We also demonstrate the usefulness of this technique to do chromosome walking in the absence of any restriction data.

The family A DNA polymerases from thermophilic bacteria are useful for PCR. The DNA polymerase from Thermus aquaticus (Taq polymerase) was the original enzyme used when practical PCR was developed, and it has remained the standard enzyme for PCR to date. Knowledge gained from structure-function relationship studies of Taq polymerase is applicable to create PCR enzymes with enhanced performance. We collected the deduced amino acid sequences of the regions from motif A to motif C in the family A DNA polymerases from metagenomic sequence data, obtained by sequencing DNAs from microorganisms isolated from various hot spring areas in Japan. The corresponding regions of the polA gene for Taq polymerase were substituted with the metagenomic DNA gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, we found an insertion sequence that affects the primer extension ability of the family A DNA polymerases. The insertion sequence is located in the finger subdomain, and it may enhance the affinity of the enzyme to DNA. Mutant Taq polymerases with the corresponding 9 amino acid insertion displayed enhanced PCR performance.

Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.