Search

We present two examples of exponential nucleic acid amplification with the polymerase chain reaction (PCR) in the presence of only one amplification primer. Cloning and sequencing of the PCR products generated by amplification of human genomic DNA revealed that the amplified sequence contained only one primer and its complement, at the two ends of the PCR product. Although these experiments were performed with primers derived from the sequence of the prostate specific antigen (PSA) gene and the normal epithelial cell‐specific 1 gene (NES1), the amplified sequences were novel and had no homology with either PSA or NES1 DNA. While both PSA and NES1 genes reside on chromosome 19q13.3‐q13.4, the amplified sequences were found by mapping to reside on chromosome 5q12 and 5p15.1‐p15.3, respectively. When we examined the mechanism of amplification by PCR using one primer in these two cases, we found that there was a high homology between the PSA primer or the NES1 primer and the two regions flanking the amplified sequence of chromosome 5q12 or 5p15. This indicated that the single PSA or NES1 primer could anneal on both strands of the DNA of that region, and mediate the exponential amplification. Since this phenomenon occurred to us twice with a limited number of different PCR reactions performed in our laboratory (< 20), we believe that it may represent a common artifact of PCR. Moreover, it appears that the palindromic primer binding sites can anneal to each other forming DNA cruciforms. J. Clin. Lab. Anal. 13:69–74, 1999. © 1999 Wiley‐Liss, Inc.

We have developed a reliable method for eliminating base-mispair amplification in selective polymerase chain reaction (PCR), which is utilized for amplifying unknown sequence fragments produced by restriction enzyme reaction. The proposed procedure applies amplified fragment length polymorphism (AFLP) with high fidelity. Selective PCR utilizes the known polymerase reaction characteristic that the complementary strand extension is strongly affected by matching a template with the 3'-terminus of the primers. However, false positive amplification is frequently observed because the specificity of terminal bases for discrimination of fragments (usually, 1-3 anchor sequences) is not enough to separate each fragment. A protocol for the selective PCR separation of every fragment was therefore investigated. A single-base mismatch was artificially introduced on the 4th base position from the 3' end of the primers to improve the hybridization specificity of anchored 2-bases at the 3' termini of primers. PCR reaction was carried out at 66 degrees C to prevent false positive amplification. The concentration of the primers having anchored-base sequences of AA, AT, TA, and TT must be three times larger than that of other primers because the Tm values for these sequences are lower than the others. As all the fragments can be separated into groups with high fidelity, the improved selective PCR will be applied to gene finding and analyzing differences on genome sequences based on AFLP.

Since its initial description over twenty years ago the PCR has become one of the most valuable and flexible tools available to biomedical research. Subsequently, refinements and modifications to the basic approach, many of which have been described in this review, have enabled the application of the PCR to many areas of diagnostic medicine and have ensured its rapid acceptance as a routine test in many pathology disciplines. The growing importance of molecular approaches to the diagnosis of disease, particularly in histopathology, will continue to secure an ever expanding role for the PCR in diagnostic pathology.

Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed.

We give a brief overview on the principles of the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative competitive PCR and real-time PCR (TaqMan technology). The literature dealing with PCR and its role in the diagnosis, pathogenesis and research of infectious diseases of the domestic cat is reviewed. Cross-contaminations which occasionally occur during handling of amplified DNA may be an important problem in the PCR laboratory. In many infectious diseases, PCR results are difficult to interpret as their predictive positive and negative values are not always known. Newer assays, such as TaqMan procedures, are becoming increasingly reliable and cost-effective. It can be expected that additional knowledge on how to interpret PCR results will soon be available.

Publisher Summary Many researches involve the analysis of multiple regions within a gene (e.g., CFTR) or the analysis of multiple loci (e.g., for human identification). Rather than perform individual polymerase chain reaction (PCR) amplification reactions for each region or locus, it is often desirable to amplify all sequences of interest simultaneously in a "multiplex" reaction. This chapter presents the optimization guidelines for multiplex PCR. There are various advantages of using multiplex PCR. It offers a significant time and cost saving benefit, especially when a large number of individuals need to be analyzed. Also, only a single aliquot of DNA or RNA is required rather than an aliquot for each marker to be analyzed. This aspect is vital for prenatal diagnosis, forensic applications, and some clinical applications in which tissue and DNA samples are repeatedly reduced. Reducing the number of tubes to which aliquots of DNA need to be added also minimizes the possibility of contamination and sample mix-up during reaction setup. It outlines the optimization strategies of multiplex PCR. Most of the guidelines and strategies used to optimize single-target PCR are similarly helpful for optimization of multiplex PCR systems. However, certain other critical factors need to be considered that are discussed in this chapter. Finally, the chapter also presents several strategies that should expedite the development of reliable multiplex amplification reactions.

Publisher Summary This chapter discusses the sequencing of polymerase chain reaction (PCR) products. PCR has proven invaluable, because it allows the amplification of specific DNA segments without having to first clone them in a microbial host. This has facilitated a wide range of applications that include mutation detection for disease diagnosis and genetic linkage studies, amplification of ribosomal genes for evolutionary studies, and environmental sampling, as well as the completion of genome sequencing projects. Of particular importance to these applications is that PCR generates DNA in quantities sufficient for sequencing. The direct sequencing of PCR products allows the generation of DNA sequence data directly from patient and environmental samples and from genomic regions that are difficult to clone. The chapter mentions that PCR sequencing can still yield high-quality sequence data. Indeed, the sequencing of PCR products is currently being used to catalog single nucleotide polymorphisms by identifying overlapping peaks in the background of otherwise high-quality sequence. If such sensitive determinations can be made by sequencing PCR products, the results obtained should be sufficient for most general sequencing applications.

A method is described for amplification of DNA fragments flanking a single known sequence that is sufficiently long to enable synthesis of a functional primer in polymerase chain reactions.

An unnatural base pair system could expand the genetic alphabet, enabling the site-specific incorporation of extra, functional components into nucleic acids and proteins. We developed an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in DNA amplification by PCR. After 20 cycles of PCR, the mutation rate of the Ds-Pn pair in an amplified DNA fragment was approximately 1%. The Ds-Pn pair system in combination with other unnatural base pairs could be useful for DNA/RNA-based biotechnology.

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

Among the different techniques used to detect oncogene amplification in tumor DNA, Southern blot and differential PCR have been the most frequently used. We report on a technical comparison of three different methods to detect gene amplification by differential PCR: ethidium bromide staining, silver staining (both after standard differential PCR), and fluorescent differential PCR. We explored the relative densitometric measure of a 119 bp fragment of the CDK4 gene versus an 82 bp fragment of the IFNG gene. In total agreement with previous studies carried out by Southern blot and differential PCR by other authors, we were able to detect CDK4 amplification in 3 of the 21 glioblastomas (14%), but only by the fluorescent differential PCR method. In conclusion, fluorescent differential PCR is more sensitive than standard differential PCR for detection oncogene amplification in tumor DNAs.

In this article we present a new concept for the detection of any specifically amplified target DNA sequences in multiple polymerase chain reactions (PCR) based on fluorescence correlation spectroscopy (FCS). The accumulation of double-stranded target DNA is monitored by the cross-correlated fluorescence signals provided by two amplification primers which are 5'-tagged with two different kinds of fluorophores (Rhodamine-Green and Cy5). Only the amplified target DNA sequence carrying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are required for determining the size and the amplified copy number of specific DNA. The PCR amplification process is started with all ingredients in a single compartment (e.g. of a microtiter plate), in which amplification and measurement are performed. This eliminates the need for post-PCR purification steps. The homogeneous one-tube approach does not depend on fluorescence energy transfer between the fluorogenic dyes. Thus, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifying DNA. The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use. For this purpose, an instrument optimized for two-color excitation and detection of two-color emission has been developed, incorporating the principle of confocal arrangement.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold™ DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2Rα, IL-2/15Rβ, γc-chain, IL-4 and IL-4Rα; IL-10, IL-15 and IL-15Rα; and IL-2, IFNγ, IL-7, IL-7Rα and IL-9Rα). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin’s lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq® DNA Polymerase, with and without manual hot-start procedure.

Human platelet antigens (HPA) can be targets for antibody responses that cause life-threatening thrombocytopenia following platelet transfusions or pregnancy. As an aid to diagnosis and prevention, serologic and DNA-based methods have been developed to type HPA. Of the DNA-based strategies, those using the polymerase chain reaction (PCR) are very sensitive, but often require processing of amplification products. Sequence-specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, current methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA-1 in a single PCR. In addition, because the absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genotype.

The amplification of target nucleic acids before hybridization is one of the most powerful approaches for the detection of low copy number RNA and DNA. The best known amplification reaction is PCR which has many applications. However, certain drawbacks of the PCR reaction provide a role for alternative amplification methods. One of these methods is the self-sustained sequence replication (3SR) reaction, which is an isothermal method for RNA amplification depending on the action of three enzymes. 3SR has been used in several in vitro applications and has also been modified for in situ use (IS-3SR). We have studied IS-3SR with the measles virus as a model and have found that it can significantly amplify the amount of intracellular RNA. Such a level of amplification could raise the amount of single copy RNA to the level of detection by conventional in situ hybridization. Although careful controls to insure its specificity must be carried out, IS-3SR has several advantages, including ease of use, preserved cell morphology, and specificity for RNA amplification, which make it an attractive alternative to the in situ PCR method.

Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun. It is suggested that betaine improves the amplification of these genes by reducing the formation of secondary structure caused by GC-rich regions and, therefore, may be generally applicable to ameliorate the amplification of GC-rich DNA sequences.

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.

The conventional methods for mRNA quantitation such as Northern blotting or ribonuclease protection assay sometimes lack enough sensitivity to study low abundance mRNAs or to work with limited amounts of biological samples. The sensitivity of the polymerase chain reaction (PCR) linked to reverse transcription (RT-PCR) has proven useful in amplifying specific mRNAs, especially those present in low copy number. Though, the quantitation of nucleic acids by means of PCR has proven problematic. The main constraint in obtaining quantitative data is inherent in the amplification reaction. Because amplification is an exponential process, small variations in the efficiency of amplification may significantly affect the final yield of the PCR product. The variables that influence the rate of the PCR include the abundance of the mRNA present in the starting material, the concentrations of the Taq DNA polymerase, dNTPs and magnesium ions, the annealing and elongation conditions, the ramping temperatures and the formation of primer secondary structures. Moreover, with the progression of the PCR cycles, reagents are consumed and inhibitors generated, leading to non-linear synthesis of DNA. Finally, tube-to-tube variations sometimes preclude accurate quantitation. Most of the above-mentioned problems can be overcome by the choice of adequate internal controls. The present report reviews two recently developed methods for RNA quantitation, the semi-quantitative PCR and the quantitative PCR illustrated for the measurement of monoamine oxidase (MAO) A and B mRNAs and the estrogen receptor (ER) mRNA respectively, with a particular emphasis on the design of appropriate internal controls to compensate for the intra- and inter-assay variability inherent to RT-PCR.

The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP-RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC-PCR) or microsatellite primed (MP-PCR) sequence, may in essence operate by the same mechanism as LP-RAPD. Using standard LP-RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air-cooled, Peltier effect, or robotic arm). LP-RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP-RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP-RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP-RAPD primers. This method has the potential to allow the production of allele-specific species markers in less than two days.

The polymerase chain reaction (PCR) has greatly enhanced the field of molecular biology by making numerous regions of the genome (coding and noncoding), in both extant and extinct taxa, accessible for detailed analysis. PCR is especially well suited for applications in systematic biology because conserved regions that flank variable portions of the genome can be used as primer sites for the amplification and sequencing of variable regions from a wide variety of species (see Palumbi 1996). In addition, PCR has been used successfully to examine polymorphisms within populations. Although many advantages are offered by PCR, the technique is not error-free. In fact, the replication-like process that drives PCR can lead to erroneously amplified products. For example, recombinant amplification products (Saiki et al. 1988; Scharf 1990) can be produced in vitro when amplifying elements of multi-gene families. Thus, the amplified product may not represent a sequence that actually exists in a single continuous stretch of DNA within any organism. Although this phenomenon was reported shortly after the development of PCR, it appears to be poorly known by many evolutionary biologists who use PCR in studies of gene evolution. Here we show that in vitro recombination also can occur when amplifying alleles of single-gene loci in heterozygous individuals, and that recombination frequency can vary as a function of using different polymerases in the extension step of PCR. In vitro recombination occurs when DNA polymerases incompletely extend a segment of DNA and the partially amplified sequence acts as a primer during subsequent amplification cycles (fig. 1). If the partially amplified segment hybridizes to an alternative form of a template strand, then subsequent PCR products will be recombinants of the two original template sequences. Partial amplification is most likely to occur when polymerases pause or prematurely disassociate from the template strand during the extension reaction, although the recombination may also be enhanced by short extension cycles or long amplifications. Although this phenomenon was initially identified in amplification of multi-gene families (Scharf, Long, and Erlich 1988; Scharf et al. 1988), we identified products of in vitro recombination in pocket gophers (Geomys) that were heterozygous for the alcohol dehydrogenase1 (Adh1) locus (Bradley et al. 1993). This experiment was designed to determine the DNA sequences of three Adh1 alleles (designated K, M, and N) that had been identified from allozyme studies. Initially, we determined the sequences of

Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature. Udgivelsesdato: 1997-Jun

Context: Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters. Aim: Optimize a cost effective PCR assay to amplify the GC-rich DNA templates. Settings and Design: Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5' untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells. Materials and Methods: A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase. Results: Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used. Conclusions: It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases.

A sex determination method has been developed using the polymerase chain reaction (PCR) which involved the amplification of the sex-determining region Y (SRY) gene and the amplification of the HLA-DQa gene as an internal control. This method, which can be applied to old and degraded DNA samples, allowed confident sexing of biological samples producing readily identifiable PCR products using two sets of primers in the one PCR reaction. The PCR products were short DNA sequences of 139bp and 239/242bp for the SRY and HLA-DQa regions respectively. The application of this method in forensic science will allow determination of the gender of perpetrators of crimes involving biological materials.

Studies of HIV molecular evolution and pathogenesis have relied on the polymerase chain reaction (PCR) to provide sequence information from infected tissues. Until recently, studies have been constrained by the limited length of fragments that can be reliably amplified. The addition of a thermostable 3′-exonuclease activity and altered cycling profiles has increased the length of target sequences that can be amplified by more than 10-fold. We have evaluated the fidelity of long PCR (LPCR). We determined that LPCR amplification maintains the distribution of sequences found in a heterogeneous sample and introduces nucleotide misincorporations at a rate comparable to that found with routine PCR. However, a significant proportion of the LPCR-amplified DNA fragments resulted from recombination events. This result suggests that LPCR amplification may have limited utility in the production and analysis of full-length HIV clones.

This chapter focuses on the polymerase chain reaction (PCR), an extremely useful technique that has revolutionized the manner in which investigators can amplify, detect, manipulate, and clone DNA fragments from a wide variety of sources. Biotechnological applications of PCR methodology include everything from improvements in basic molecular biology methods to clinical uses in the diagnostic detection of genetic mutations, viral infections, or molecular “fingerprinting” as applied to forensic medicine. Several factors influence the fidelity and efficiency of the PCR process. The concentration of various components constituting a typical PCR is a primary determinant of successful amplification. These include the concentration of Thermus aquaticus ( Taq ) polymerase, deoxynucleotide triphosphates, magnesium ions, template DNA, and primers in the reaction. The temperature and times of each step within each cycle and total number of cycles can dramatically affect product yield and specificity. The differential effects of temperature and salt concentration in nucleic-acid hybridization is called “stringency” and this is important in discerning appropriate conditions for PCR. The availability of a purified heat-stable DNA polymerase from the thermophilic bacterium Taq polymerase greatly facilitates the enzymatic catalysis of DNA amplification.

Publisher Summary The more recent development of the polymerase chain reaction (PCR) enables the amplification of target DNA in the specimen, increasing the sensitivity of the tests undertaken for diagnosis of infections by several orders of magnitude. Positive hybridization data, and in the case of PCR, demonstration in gels of the amplified target sequence, may suffice to indicate the presence of the infectious agent. The first DNA probes applied to diagnosis of mycoplasma infections consisted of cloned ribosomal RNA genes, such as the recombinant plasmid pMC5. Another class of DNA probes consisting of chromosomal segments specific for a certain mollicute species is discussed. These segments are derived from a genomic library of the specific mycoplasm. PCR tests are several orders of magnitude more sensitive than those based on direct hybridization with a DNA probe. The first major issue in the development of a PCR-based test concerns the selection of the appropriate target sequences for amplification. The sequence to be amplified can be chosen from a published mycoplasmal gene sequence or from a randomly cloned DNA fragment demonstrated to be specific for the mycoplasma to be detected. The methodology of target sequence amplification and optimization of the PCR test conditions as well as the identification of the PCR products are presented.

High-annealing-temperature (HAT) primers for polymerase chain reaction (PCR) were designed and tested. These HAT primers were used to develop an amplification procedure that employs hypervariable extension times in combination with two temperature amplification cycles, master mixes and hot start to achieve highly reproducible, low-copy-number PCR amplification. The employment of HAT primers drastically reduced cycling times and essentially eliminated nonspecific amplification products even in the presence of vast excesses of nonspecific DNA sequences. Hypervariable extension times provided a simple, noninvasive approach for dynamically modifying the reaction environment during amplification. In addition, this procedure makes extremely efficient use of enzyme, reducing the amount of Taq or Replitherm polymerase to 0.4 and 0.12 U per 50 μl reaction, respectively. The use of PCR master mixes increased the reproducibility and portability of the assay. And finally, a modified form of hot start was employed to reduce primer oligomer formation. HAT primers were designed for the amplification of human cytomegalovirus (HCMV). These primers were used to develop a highly specific, low-copynumber PCR assay that employed a combined annealing/extension temperature of 70 °C or 72 °C. Nonspecific bands, other than primer oligomer bands, were not detected even in the presence of a vast excess of human genomic DNA. These primers can be employed to calibrate the temperature on different thermocylcers as well as to take into account environmental factors that influence amplification. This would be especially advantageous when porting protocols to different machines and simplifying the use of PCR in field studies as well as for identifying factors that influence the local stability at the 3′ end of primers.

Publisher Summary This chapter describes PCR strategies that have successfully been used to isolate novel cytochrome P450 gene sequences from petunia. Most of the plant P450 genes of known function have been isolated via protein purification strategies except for flavonoid 3',5'-hydroxylase genes that have been isolated via a polymerase chain reaction (PCR) amplification strategy. The chapter shows that PCR is a very powerful method for isolating large numbers of novel P450 gene sequences. It is often desirable to isolate specific P450 genes rather than all P450 genes expressed in a particular organ or tissue. The PCR approach can be modified in a number of ways to clone P450 genes that are expressed in one tissue (or genotype) but not in another. It is unlikely that one pair of PCR primers will allow amplification of every P450 gene, but the use of a few different primer combinations will amplify most of them. The most difficult step lies in the identification of gene function of a newly isolated gene. However, the availability of a genetic map and/ or genetically defined mutants of known P450 genes can facilitate the identification of gene function.

The protocol described in this chapter has been used to detect Mycoplasma pneumoniae and M . genitalium in clinical specimens. The polymerase chain reaction (PCR) parameters to be optimized include the composition of the mixture, the volume used, and the amplification cycle. Standard PCR protocols can be used for the amplification of most DNA target sequences. The PCR-amplified fragment is visualized on an electrophoresis gel by ethidium bromide staining. The choice of gel, agarose or acrylamide, depends on the fragment size. Several DNA molecular weight markers suitable for approximate size analysis of PCR products are commercially available. The chapter illustrates concentration of different components, such as enzyme concentration and deoxynucleotide triphosphates. In routine assays, a direct blot of PCR products (dot or slot blot) may be advantageous. This procedure requires no electrophoresis and no DNA transfer, but the drawback is that the PCR product size cannot be visualized.

Positive identification of human remains has always been one of the main goals of Forensic Science. Fingerprints, dental or skeletal data were considered useful tools for this purpose until now (Farinelli 1993; Sopher 1993). However, the application of molecular biology methods in forensic laboratories has achieved new perspectives in the direct identification by resolving cases with critical amount of sample for analysis (Hagelberg 1991; Sullivan 1992). This can be made through the study of inherited characters in parents or relatives and direct matching with human remains samples. In this comunication we show the results in two cases received in the Servicio de Analitica de la Comisaria General de Policia Cientifica, Madrid (Spain). In one of them, human remains were buried over four years, in the other they suffered the effect of high temperatures because of a fire. These two cases were selected because they express critical factors in the necroidentification.

Many groups have now published data based on the in situ detection of PCR-amplified DNA and cDNA. As with standard in situ hybridization or PCR, variables that can affect in situ PCR results include type of fixative and time of fixation, protease digestion, and the composition of the amplifying solution and oligoprobe cocktail. Investigators new to the field of in situ PCR should first try direct incorporation of the reporter molecule into paraffin-embedded tissue sections. Although nonspecific DNA synthesis is generated under these conditions, one can develop the confidence of synthesizing DNA inside the nucleus and appreciate the importance of protease digestion time to successful RT in situ PCR. It is an arguable statement that the in situ detection of PCR-amplified DNA and cDNA will have a very strong impact on many diverse fields, such as oncogenesis, embryology, RNA trafficking, and detection of viral diseases, as it already has on our understanding of the pathogenesis of HIV-1 infection.

The polymerase chain reaction (PCR) is firmly established as the method of choice for DNA amplification, though alternative strategies, such as the ligase chain reaction, may also be employed. Despite the continued development of PCR applications for gene mapping and diagnostics, few revolutionary improvements have been made to the technique. The major exception is long-accurate PCR, which has increased the length of amplifiable DNA by an order of magnitude.

In most studies, PCR applications have been coupled with either a gel electrophoresis step or liquid/solid support hybridization (see chapter 2.5). Many detection methods are very time consuming or their sensitivity is too low [1].

In routine casework, laboratories performing genotyping in forensic applications have often to deal with samples containing little and/or badly preserved DNA. We present here the results obtained in our laboratory under such difficult situations.

Publisher Summary This chapter discusses the use of polymerase chain reaction (PCR) in quantitative applications. Although the sensitivity of PCR has rendered it the method of choice in the detection of low amounts of nucleic acid target, its utility as a quantitative tool has been slower in gaining credibility. The ability of PCR to make rare sequences detectable also makes it extremely challenging to extrapolate the amounts of initial input template molecules from the measured amounts of product molecules after many cycles of amplification. In the past few years, a number of PCR strategies based on different methodological and technical approaches have been adopted for the semiquantitative or quantitative analysis of nucleic acids. These strategies deal with the basic problem of making the amplification process as predictable as possible. Because of the exponential nature of PCR, even minor variations in efficiency can lead to large deviations at the end of the reaction that will confound the quantitative relationship between the product and initial template. An understanding of the theoretical and technical aspects of the kinetics of PCR product accumulation is imperative to designing quantitative PCR experiments.

Publisher Summary The polymerase chain reaction (PCR) is an in vitro method that enables the exponential enzymatic synthesis of a targeted segment of DNA. Thus, PCR provides a rapid, highly sensitive, and specific means of nucleic acid detection and isolation. Direct sequencing of DNA produced by a PCR has consequently received much attention of late. However, there are a number of difficulties associated with the direct sequencing of PCR products. These include the presence of nonspecific amplification products and the excess primers and deoxyribonucleoside triphosphates carried over from the amplification to the sequencing reaction. In addition, the small size of the DNA template in relatively high concentration results in rapid reannealing of the double-stranded DNA. Numerous methods have been described that attempt to overcome the inherent difficulties associated with the sequencing of double-stranded PCR products. Because of the simplicity associated with the direct sequencing of double-stranded DNA, a protocol has been developed to overcome the problem of the reassociation of template DNA and the consequent reduction of signal intensity. The protocol uses chemiluminescent detection, which has several advantages over radioactive detection methods.

Publisher Summary Polymerase chain reaction (PCR) has become an extremely useful technique in molecular biology. The chapter describes PCR mutagenesis protocol that gives rise to multiple point mutations. The total number of sites varied in a mutagenic oligo dictated the number of point mutations introduced in the final product obtained as the predominant species. In the case of BS II and BS III, because there are three or more mixed sites in oligos, there is a preponderance of clones with three or more point mutations. It is possible that during the synthesis of mixed-site oligos, the oligos with multiple mutations were disproportionately abundant over oligos with single point mutations. At the annealing temperature of the PCR (45–50°), the mutagenic oligos with double or more mutations could form more stable hybrids with the mutant amplified DNA, produced within a few cycles of the first PCR containing a single shared mutation, than with the wild-type template. Further amplification resulting from this stable annealing would give rise to mutant DNA with multiple mutations in subsequent cycles. In addition, the method described in the chapter is well suited for introducing multiple mutations in a defined region. Although the transformants need to be screened by DNA sequence analysis in this procedure, different mutants can be identified by a single sequencing gel. Sequencing the transformants also achieves the purpose of verifying the accuracy of the PCR amplification, as would be required for any mutagenesis protocol.

PCR amplification is a powerful technique for cloning related genes. Compared to traditional structure-based methods, it is faster and more specific. When properly designed and executed, the isolation of related genes by PCR amplification can be accomplished in less than 1 week. The key element to success is the proper choice of PCR primers, which are designed according to the degree of conservation of amino acid sequence in the protein(s) of interest and the degeneracy of the resulting oligonucleotides. We demonstrate the utility of this approach for the isolation of PTP genes from distantly related organisms.

This chapter focuses on the detection of viral pathogens using polymerase chain reaction (PCR) amplification. PCR is a relatively simple procedure consisting of repetitive cycles of oligonucleotide primer-directed DNA replication. A selected genetic target is copied in a three-step reaction: denaturing the target DNA, annealing, and extending the bound primers by DNA polymerase action. The use of Taq DNA polymerase resulted in increased specificity of PCR amplification reactions by permitting higher primer annealing and polymerase extension temperatures to be used, decreasing the amount of non-specific target amplification. The high analytical sensitivity of PCR-based procedures facilitates the detection of minute amounts of pathogens in biological specimens that are undetectable using more conventional technology. The use of uracil-N-gylcosylase in PCR protocols improves the sensitivity of PCR amplifications by degrading nonspecific extension products that result from low-stringency primer binding that occurs below the optimal primer annealing temperature. Direct detection of viral particles or nucleic acid is the most reliable method for diagnosis of human immunodeficiency virus (HIV) infection in newborns of HIV-infected women.

This chapter describes an isothermal method for amplification of nucleic acids based on ligation of a promoter sequence to a target nucleic acid, resulting in target amplification through a transcribed RNA intermediate. This target amplification methodology has been combined with solution hybridization based enzymatic detection of amplified target nucleic acid to produce a highly sensitive method for the detection of nucleic acids. Application of these technologies to detection of human papillomaviruses (HPV) is described. The widespread utility of amplification in molecular biology and molecular diagnostic procedures has stimulated the development of other nucleic acid amplification methods. Some of these methods are dependent on the presence of a DNA target and amplify a target sequence; other methods are based on replicatable probes that are amplified after hybridization to reveal the presence of the probe. The ligation-activated transcription reaction is performed under isothermal conditions without thermocycling. The reaction is capable of using RNA or DNA as the starting template and produces both single-stranded RNA and DNA amplification products.

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.

Publisher Summary Polymerase chain reactions (PCR) are often performed in searches for the genes of interest, or their messenger RNA transcripts. However, the direct amplification and sequencing of complementary DNA (cDNA) products that encompass 3' and 5' ends have been problematic. The oligo(dT) primer that is traditionally used in the cDNA synthesis step can anneal at any position along the length of a natural (3') or appended (5') poly(A) tail. Subsequently, PCR products of a single, discrete size are not obtained. To enable the acquisition of discrete, first-round PCR products, a “lock-docking” cDNA synthesis primer and the accompanying PCR procedure are described in the chapter. These PCR products can be directly sequenced. This lock-docking primer system may be applied to a novel method for searching previously unidentified gene family members. While PCR targets in the internal, coding regions tend to be of similar lengths, targets in the 3' regions tend to be of varying lengths. Thus, internal PCR products are difficult to separate for further study, while products from 3' regions may be separated by agarose gel electrophoresis.

Publisher Summary Formamide low-temperature polymerase chain reaction (FoLT PCR) is a rapid PCR protocol that allows direct amplification of DNA from whole blood and tissues without any preparative steps—that is, the raw material can be put directly into the tube containing the PCR mixture. It entails the use of 18% formamide, reduced incubation temperatures (80°, 40°, and 60°C), and Thermus thermophilus (Tth) DNA polymerase. This technique has been applied to amplify histocompatibility genes and enzyme genes in several species, including mice and humans. Blood and solid tissues have been used successfully. This chapter demonstrates its use in screening transgenic mice. FoLT PCR saves considerable time and is economical when compared with competing methodologies. Another important characteristic of the FoLT PCR protocol is that even though it is a rapid direct method, it does not compromise sensitivity as compared to protocols that involve DNA purification. One of the advantages of FoLT PCR is that it is suitable for use with most widely used anticoagulants. The only anticoagulants that are not recommended for routine use are lithium heparin, fluoride heparin, and sodium citrate.

A laboratory centrifuge is a piece of laboratory equipment, driven by a motor, which spins liquid samples at high speed. There are various types of centrifuges, depending on the size and the sample capacity. Like all other centrifuges, laboratory centrifuges work by the sedimentation principle, where the centripetal acceleration is used to separate substances of greater and lesser density. The information for synthesizing the molecules that allow organisms to survive and replicate is encoded in genomic DNA. Extracted and purified genomic DNA is very important for the analysis of single nucleotide polymorphisms (SNPs), disease states, and for many other multiplex and real-time PCR applications. The aim of this work is to design a genomic DNA extraction system that satisfies downstream application needs necessary for the successful completion of experiments and able to isolate a purified genomic DNA from many sources from bacteria to humans and also is able to encompass tissues from blood to muscle and from leaf to seed. Results revealed that the proposed system works with high efficiency and spins at up to 12,000 RPM to facilitate separation of the different phases of the extraction.

We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called ‘helper-dependent chain reaction’. The process uses disabled primers called ‘drivers’ that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA., Funding for open access charge: Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. National Health & Medical Research Council