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This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a programme to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with GC or AT skew, of CG content and purine-pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyses sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and online version at http://primerdigital.com/tools/pcr.html.

Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

Multiplex PCR allows the simultaneous amplification of up to dozens of target fragments in a single PCR. It is therefore a powerful tool to obtain many kilobases of continuous sequence from minute amounts of ancient DNA (aDNA), which usually must be amplified in multiple short and overlapping fragments. Because significantly less template is required compared to amplifying each fragment separately, multiplex PCR is particularly beneficial when the fossil material itself, or access to the fossil material, is limited. The recently refined two-step multiplex PCR protocol consists of a first-step reaction (the actual multiplex PCR) that then acts as the template for the second-step PCR. During the second step, nested primers are used in individual amplification reactions. Although the same set of primers can be used in both steps, using a nested set in the second step adds an additional level of selectivity and specificity, minimizing PCR artifacts. This is particularly important when complex mixtures of template DNA, such as aDNA extracts, are amplified.

PCR amplification of DNA is routine in modern molecular biology. However, the application of PCR to ancient DNA (aDNA) experiments often requires significant modification to standard protocols. The degraded nature of most aDNA fragments requires targeting shorter fragments, performing replicate amplifications, incorporating multiple negative controls, combating PCR inhibition, using specific DNA polymerases to deal with damaged bases, working in a separate aDNA facility, and modifying the PCR recipe to deal with damaged and low copy-number target DNA. In this chapter, we describe how and why these procedures are implemented, discuss aDNA-specific troubleshooting methodology, and suggest modifications to commercial cloning and sequencing procedures to reduce the expense of PCR product cloning.

In silico PCR analysis is a useful and efficient complementary method to ensure primer specificity for an extensive range of PCR applications from gene discovery, molecular diagnosis, and pathogen detection to forensic DNA typing. In silico PCR, SNPCheck, and Primer-BLAST are commonly used web-based in silico PCR tools. Their applications are discussed here in stepwise detail along with several examples, which aim to make it easier for the intended users to apply the tools. This virtual PCR method can assist in the selection of newly designed primers, identify potential mismatches in the primer binding sites due to known SNPs, and avoid the amplification of unwanted amplicons so that potential problems can be prevented before any "wet bench" experiment.

Proofreading DNA polymerase fusions offer several advantages for long-range PCR, including faster run times and higher fidelity compared with Taq-based enzymes. However, their use so far has been limited to amplification of small to mid-range targets. In this article, we present a modified protocol for using a DNA polymerase fusion to amplify genomic targets exceeding 20 kb in length. This procedure overcomes several limitations of Taq blends, which up until recently, were the only option for long-range PCR. With a proofreading DNA polymerase fusion, high-molecular-weight amplicon can be generated and analyzed in a single day, and a significant proportion is expected to be error-free.

Multiplex Polymerase Chain Reaction (PCR) can be used for the simultaneous detection of plant viruses. Multiple primer pairs or polyvalent primer pairs can be used to detect and identify several viruses in a single PCR.

The development of instruments that allow real-time monitoring of fluorescence within PCR reaction vessels is a significant advance in clinical bacteriology. The technology is very flexible, and many alternative instruments and fluorescent probe systems are currently available. Real-time PCR assays can be completed very rapidly, because no manipulations are required after amplification. Identification of amplification products by probe detection in real time is highly accurate compared with size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Finally, probe melting analysis can detect sequence variants including single base mutations.

The polymerase chain reaction (PCR) has revolutionized many areas of medicine, including hemostasis. Although this volume is not devoted to PCR, many of the chapters employ the technique at some point to amplify specific DNA or RNA sequences. This chapter, therefore, provides a brief outline of the techniques and methods for the amplification of both DNA and RNA. It should be remembered that no single protocol will be suitable for all PCR reactions and that optimization of the reaction is necessary whenever a new PCR reaction is undertaken. Further information on PCR can be found in one of the many textbooks on the subject that are available.

Quantitative polymerase chain reaction (PCR) is aimed to determine the absolute or relative amounts of RNA or DNA sequences in a given sample. There are two facts limiting the convenience of this approach. First, in most cases, only one or two sequences are amplified in a given round of amplification. If a family of sequences are to be quantitated, as many amplification reactions are necessary. However, it has been shown that complex populations could be amplified in a sequential independent way (1-3). A major concern about the amplification of whole populations are the biases for or against some sequences. In fact, it appears that these biases are not important and that the amplified populations are quite representative of the original mixture of sequences (1,4). This makes possible a score of PCR applications such as differential display analysis (5) or representational difference analysis (6), which are aimed to detect qualitative and quantitative differences between sequences present in genomes or messenger RNA (mRNA) populations. This also implies that it is possible to measure the amount of numerous sequences in the amplicons.

The polymerase chain reaction (PCR) approach to chromosome assignment consists of analyzing the profile of specific amplification products of the gene of interest using genomic DNAs from a panel of somatic cell hybrids as templates (see Fig 1). Each hybrid somatic cell line contains the normal complement of chromosomes of one species, along with one or more additional chromosomes from a second species (1,2). The PCR approach has obvious advantages over the conventional Southern blotting method of chromosome assignment, including speed, sensitivity, and conservation of the genomic DNAs, which are difficult and labor intensive to generate.

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.

The association of c-erbB-2 oncogene amplification and prognostic factors was intensively studied in human gynecological carcinomas, especially in mammary carcinoma (1). Positive lymph nodes, estrogen and progesterone receptor negative tumors, and short survival time correlate with c-erbB-2 amplification.

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic acid probes are used in a screening process to check whether or not a plaque or a colony contains the sequence of interest. There have been attempts to isolate specific DNA fragments using immobilized DNA, in which particular DNA fragments were enriched by hybrid selection and then the concentrated library was screened by a specific DNA probe (1,2). Recently, polymerase chain reaction (PCR) has been applied to the cloning of genes. Friedmann et al. (3) first used PCR to screen λgt11 library with two gene-specific primers. This protocol can be effectively used to isolate a particular DNA fragment between two specific primers or to generate nucleic acid probe from cDNA libraries. The unknown sequences flanking the fragment between the two specific primers cannot be amplified by this method.