A positive control can also be a known positive sample, which is usually a substitute for an absolute standard and used only to test for the presence or absence of a target. A no RT control, where real-time RT-PCR is carried out without reverse transcriptase, should be included when performing gene expression analysis. For viral load monitoring, a no RT control may be necessary, depending on the sample type and the life cycle of the virus species detected.
Since reverse transcription cannot take place, a no RT control reaction allows detection of contaminating DNA, such as DNA from viral sequences integrated into the host genome. An internal, positive control can be used to test for the presence of PCR inhibitors. A duplex reaction is carried out, where the target sequence is amplified with one primer—probe set, and a control sequence i.
The internal, positive control should be at a high enough copy number for accurate detection. If the internal, positive control is detected, but the target sequence is not, then this indicates that the amplification reaction was successful and that the target sequence is absent or at too low a copy number to be detected.
Several factors can generate a false negative result, such as errors in sample extraction or thermocycler malfunction. The most practical approach to control for the presence of inhibitors is to include an Internal Positive Control, or Internal Control IC.
This IC is simultaneously extracted and amplified or only amplified in the same tube with the pathogen target, and should always be combined with an external positive control to prove the functionality of the reaction mix for amplification of the target. This combination rules out inhibition, among other malfunctions, and confirms that a negative result is truly negative.
Not all internal controls are the same see table Features of internal controls , and each IC concept has value for specific applications. Endogenous ICs occur naturally in test specimens, such as a sequence of the host genome e.
Exogenous ICs, on the other hand, are spiked into samples either during nucleic acid extraction or before PCR amplification. Exogenous ICs can be homologous, where an artificial template is constructed with the same primer binding sites as the targeted pathogen sequence.
Although the same primer set is used for target and IC, the sequence differs, enabling differentiation of pathogen and IC amplicons with different probes. Heterologous ICs, on the other hand, are designed with their own primers and probe. Endogenous and exogenous homologous ICs carry the risk of impairing detection sensitivity for the pathogen target due to competition for reaction components. For example, a high starting amount of an endogenous IC template can impair assay sensitivity.
This high starting amount can result from variations in the sample type or sampling technique. In the case of RNA applications, the high starting amount can also be due to enhanced expression levels of the IC due to disease-related cellular pathology. In the case of exogenous homologous ICs, using the same primers to amplify both target and IC leads to primer competition. Additionally, both endogenous and homologous ICs involve tedious IC design, and their use is restricted to a few applications or even individual assays.
In the context of process safety and workflow simplification, exogenous heterologous ICs are the most informative and flexible. The amount of IC template spiked into a sample is defined and consistent, and unrestricted design options enable optimization of IC properties.
Only heterologous ICs allow for a design and setup that prevents competition for PCR components, and heterologous ICs are suited as universal controls, thereby making their implementation in new assays easy.
Multiplex PCR employs different primer pairs in the same reaction for simultaneous amplification of multiple targets. This type of PCR often requires extensive optimization of annealing conditions for maximum amplification efficiency of the different primer—template systems and is often compromised by nonspecific PCR artifacts.
A stringent hot-start procedure and specially optimized buffer systems are absolutely crucial for successful multiplex PCR. Compared with standard PCR systems using only 2 primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs.
Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products.
Commercial PCR kits are available that are specifically designed to overcome the challenges of multiplex PCR and it is recommended that, where possible, such a kit is used.
However, amplification of PCR products longer than 4 kb often fails without lengthy optimization. Reasons for failure include nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions — all factors which have a greater effect on the amplification of longer PCR products than on shorter ones.
DNA damage during PCR cycling can be minimized with specific buffering substances that stabilize the pH of the reaction. Commercial PCR kits are available that are specifically designed to overcome the challenges of long-range PCR, for example, by using an optimized mixture of Taq DNA polymerase and proofreading enzymes, and it is recommended that, where possible, such a kit is used.
Single-cell PCR provides a valuable tool for genetic characterization using a limited amount of starting material. By flow cytometry or micromanipulation, individual cells of interest can be isolated based on cell-surface markers or physical appearance. Amplification of low-abundance template molecules — which can be as low as one or two gene copies — requires a PCR system that is highly efficient, specific, and sensitive.
Faster PCR amplification enables increased PCR throughput and allows researchers to spend more time on downstream analysis. The demand for reducing time-to-result is met by the recent development of faster PCR techniques. Fast PCR can be achieved using new thermal cyclers with faster ramping times or through innovative PCR chemistries that allow reduced cycling times due to significantly shortened DNA denaturation, primer annealing, and DNA extension times.
Fast-cycling PCR reagents must be highly optimized to ensure amplification specificity and sensitivity. The method requires two sets of primers to be designed: one set that anneals to unchanged cytosines i. Amplification products derived from the primer set for unchanged sequences indicates the cytosines were methylated and thus protected from alteration 6. Stringent and highly specific PCR conditions must be used to avoid nonspecific primer binding and the amplification of PCR artifacts.
This is particularly important as the conversion of unmethylated cytosines to uracils reduces the complexity of the DNA and increases the likelihood of nonspecific primer—template binding. See High-fidelity DNA polymerase for more information. It uses small, nonspecific primers to amplify seemingly random regions of genomic DNA.
Successful primer pairs produce different banding profiles of PCR products between individuals, strains, species, etc. It is often used for cloning the remainder of incomplete cDNAs. There are two techniques:. In situ PCR allows cellular markers to be identified and further enables the localization to cell-specific sequences within cell populations, such as tissues and blood samples.
Therefore, it is a powerful tool in applications such as the study of disease progression. Fresh or fixed cells or tissue samples can be used in the procedure, although preparation of the sample is critical to the result, with fixation having a direct influence on PCR signal.
The procedure is suitable for use with radiolabeled, fluorescently labeled or biotin-labeled nucleic acid probes. After reverse transcription and amplification, amplified products are visualized using gel electrophoresis. The banding patterns observed can be compared to identify differentially expressed cDNAs in the 2 populations.
Invented in the s, the technique fast became a key tool in gene expression analysis. When performing real-time RT-PCR, the primers and the enzyme for reverse transcription must be carefully chosen.
The primers should allow reverse transcription of all targets of interest, and the reverse transcriptase should yield cDNA amounts that accurately represent the original RNA amounts to ensure accurate quantification. In addition, the effects of the components of the RT reaction on subsequent real-time PCR must be minimized.
An aliquot of the reverse-transcription reaction is then added to the real-time PCR. It is possible to choose between different types of RT primers, depending on experimental needs. Use of oligo-dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from a single RT reaction.
In addition, precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage. The fast procedure enables rapid processing of multiple samples and is easy to automate. The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required.
The choice of primers for reverse transcription depends on whether one-step or two-step RT-PCR is being carried out. If oligo-dT primers are used, only mRNAs will be reverse transcribed starting from the poly-A tail at the 3' end. Since reverse transcription is initiated from several positions within the RNA molecule, this will lead to relatively short cDNA molecules.
In comparison, gene-specific primers allow reverse transcription of a specific transcript. A universal priming method for the RT step of real-time two-step RT-PCR should allow amplification and detection of any PCR product regardless of transcript length and amplicon position, and achieve this with high sensitivity and reproducibility. We recommend testing dilutions of the RT reaction in real-time PCR to check the linearity of the assay. This helps to eliminate any inhibitory effects of the RT reaction mix that might affect accurate transcript quantification.
Alternatively, the reverse transcriptase can skip over looped-out regions of RNA, which are then excluded from the synthesized cDNA. RNase H digestion has been previously shown to improve RT-PCR yield and to be required for amplification of some sequences, even as short as bp 7.
A critical factor in RT-PCR is the selection of appropriate primers for maximal efficiency and specificity. Primer specificity is affected by a number of factors, including sequence, primer location, and the RT-PCR system used. Even the comb binding of the book makes it suitable for bench-top use The breadth of the applications and methodology described in this book should give even the novice the confidence and skill to make PCR work The 30 or so applications articles give the experienced reader a good overview of the power and utility of PCR.
This book is an excellent compilation for all who, having a general background in molecular biology, wish to practice PCR No user of the PCR technique could wish to miss it. They are written by researchers who use PCR successfully in their laboratory and who do not shy away from pointing out the potential pitfalls. This extremely useful publication of PCR Protocols goes far beyond the simple amplification of nucleic acids A well-organized index is provided.
This book will make it more difficult to find an excuse for not using the PCR technique. White, A c a d e m i c Press, Salunkhe, N. Bhat and post-harvest losses that o c c u r along ditional notes are given o n potential B.
Desai, Springer-Verlag, Salunkhe, difficulties or mistakes, and indica- DM Related Papers. Addition of betaine, DMSO and formamide can be helpful when amplifying GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerases to stall.
GC-rich templates can be problematic due to inefficient separation of the two DNA strands or the tendency for the complementary, GC-rich primers to form intermolecular secondary structures, which will compete with primer annealing to the template. Betaine reduces the amount of energy required to separate DNA strands Rees et al. DMSO and formamide are thought to aid amplification in a similar manner by interfering with hydrogen bond formation between two DNA strands Geiduschek and Herskovits, In some cases, general stabilizing agents such as BSA 0.
These additives can increase DNA polymerase stability and reduce the loss of reagents through adsorption to tube walls. Ammonium ions can make an amplification reaction more tolerant of nonoptimal conditions. It is important to minimize cross-contamination between samples and prevent carryover of RNA and DNA from one experiment to the next. Use separate work areas and pipettors for pre- and post-amplification steps.
Use positive displacement pipettes or aerosol-resistant tips to reduce cross-contamination during pipetting. Wear gloves, and change them often. There are a number of techniques that can be used to prevent amplification of contaminating DNA. PCR reagents can be treated with isopsoralen, a photo-activated, cross-linking reagent that intercalates into double-stranded DNA molecules and forms covalent, interstrand crosslinks, to prevent DNA denaturation and replication.
These inter-strand crosslinks effectively render contaminating DNA unamplifiable. For UNG to be an effective safeguard against contamination, the products of previous amplifications must be synthesized in the presence of dUTP.
Since dUTP incorporation has no noticeable effect on the intensity of ethidium bromide staining or electrophoretic mobility of the PCR product, reactions can be analyzed by standard agarose gel electrophoresis. While both methods are effective Rys and Persing, , UNG treatment has the advantage that both single-stranded and double-stranded DNA templates will be rendered unamplifiable Longo et al. Procedures for creating and maintaining a ribonuclease-free RNase-free environment to minimize RNA degradation are described in Blumberg, The use of an RNase inhibitor e.
The most commonly used DNA polymerases for PCR have no reverse transcriptase activity under standard reaction conditions, and thus, amplification products will be generated only if the template contains trace amounts of DNA with similar sequences.
Figure 3. Amplification of a specific message in total RNA. The specific bp amplicon is indicated. Selection of an appropriate primer for reverse transcription depends on target mRNA size and the presence of secondary structure. Random hexamers prime reverse transcription at multiple points along the transcript.
For this reason, they are useful for either long mRNAs or transcripts with significant secondary structure. Whenever possible, we recommend using a primer that anneals only to defined sequences in particular RNAs sequence-specific primers rather than to entire RNA populations in the sample e. To differentiate between amplification of cDNA and amplification of contaminating genomic DNA, design primers to anneal to sequences in exons on opposite sides of an intron so that any amplification product derived from genomic DNA will be much larger than the product amplified from the target cDNA.
This size difference not only makes it possible to differentiate the two products by gel electrophoresis but also favors the synthesis of the smaller cDNA-derived product amplification of smaller fragments is often more efficient than that of long fragments.
Regardless of primer choice, the final primer concentration in the reaction is usually within the range of 0. The higher reaction temperature will minimize the effects of RNA secondary structure and encourage full-length cDNA synthesis. It has been reported that AMV reverse transcriptase must be inactivated to obtain high yields of amplification product Sellner et al. Most RNA samples can be detected using 30—40 cycles of amplification.
If the target RNA is rare or if only a small amount of starting material is available, it may be necessary to increase the number of cycles to 45 or 50 or dilute the products of the first reaction and reamplify.
Thermostable DNA polymerases revolutionized and popularized PCR because of their ability to withstand the high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing. These two groups have some important differences. When the amplified product is to be cloned, expressed or used in mutation analysis, Pfu DNA polymerase is a better choice due to its high fidelity. However, for routine PCR, where simple detection of an amplification product is the goal, Taq DNA polymerase is the most commonly used enzyme because yields tend to be higher with a nonproofreading DNA polymerase.
The single-nucleotide overhang can simplify the cloning of PCR products. The fidelity is slightly higher at lower pH, lower magnesium concentration and relatively low dNTP concentration Eckert and Kunkel, ; Eckert and Kunkel, For products larger than approximately 10kb, we recommend an enzyme or enzyme mix and reaction conditions that are designed for long PCR.
This enzyme is commonly used in PCR Gaensslen et al. The error rate of Tth DNA polymerase has been measured at 7. Tth DNA polymerase can amplify target DNA in the presence of phenol-saturated buffer Katcher and Schwartz, and has been reported to be more resistant to inhibition by blood components than other thermostable polymerases Ehrlich et al.
Pfu DNA polymerase can be used alone to amplify DNA fragments up to 5kb by increasing the extension time to 2 minutes per kilobase. However, the proofreading activity can shorten PCR primers, leading to decreased yield and increased nonspecific amplification. Some DNA-dependent DNA polymerases also possess a reverse transcriptase activity, which can be favored under certain conditions.
However, for shorter templates with complex secondary structure, AMV reverse transcriptase may be a better choice because it can be used at higher reaction temperatures. As the names suggest, the deletion mutant had a specific sequence in the RNase H domain deleted, and the point mutant has a point mutation introduced in the RNase H domain. The point mutant is often preferred over the deletion mutant because the point mutant has DNA polymerase activity comparable to that of the wildtype M-MLV enzyme, whereas the deletion mutant has a slightly reduced DNA polymerase activity compared to that of the wildtype enzyme Figure 4.
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Life Sciences Cell Biology. Methods in Molecular Biology Free Preview. Show next edition.The protocoks of the polymerase chain applicatoins PCR by K. Mullis and co-workers in revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation, and cancer research, are challenging today's PCR technologies with more demanding requirements. These include the need for guife throughput, higher assay sensitivity, and reliable data analysis. Assay development and evaluation, reproducibility of data, and time to result are still major problems encountered by researchers. PCR amplification is performed routinely and thousands of PCR protocols have been developed, yet researchers still encounter technical difficulties with PCR experiments and often fail to obtain specific amplification products. Although there are proocols different challenges e. 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