PCR Amplification. The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment.

However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of different types of PCR methods, applications and optimization. A detailed treatment of these methods is beyond the scope of this publication. However, an extensive bibliography is provided in the References section for researchers who require more comprehensive information. The PCR process was originally developed to amplify short segments of a longer DNA molecule (Saiki et al. A typical amplification reaction includes target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (d.

NTPs), reaction buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. Bruel Kjaer North America. This series of temperature and time adjustments is referred to as one cycle of amplification.

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Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 2. Each cycle of PCR includes steps for template denaturation, primer annealing and primer extension (Figure 1. The initial step denatures the target DNA by heating it to 9. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single- stranded DNA template for replication by the thermostable DNA polymerase.

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In the next step of a cycle, the temperature is reduced to approximately 4. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 1. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 7. The extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 9.

If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. After 2. 0–4. 0 cycles, the amplified product may be analyzed for size, quantity, sequence, etc., or used in further experimental procedures.

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An animated presentation illustrating the PCR process is available. Additional Resources for Basic PCRTechnical Bulletins and Manuals. TB2. 54 Go. Taq. Reprod. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome- specific target for gender assignment and a bovine- specific satellite sequence as a control. PCRs were performed using Go.

Taq. Cell Sci. 12. As part of the study, RT- PCR was used to detect the presence of other neurotrophin receptors in their model cell line SW4.

Reverse transcription was performed using the Reverse Transcription System and 1. The resulting c. DNA (5. RT- PCR results were confirmed by Western blot analysis. Pub. Med Number: 1.

Thermostable DNA polymerases used for basic PCR require a DNA template, and as such, the technique is limited to the analysis of DNA samples. Yet numerous instances exist in which amplification of RNA would be preferred. To apply PCR to the study of RNA, the RNA sample must first be reverse transcribed to c. DNA to provide the necessary DNA template for the thermostable polymerase (Figure 1. This process is called reverse transcription (RT), hence the name RT- PCR. Avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M- MLV or Mu.

LV) reverse transcriptases are generally used to produce a DNA copy of the RNA template using either random primers, an oligo(d. T) primer or sequence- specific primers. Promega offers Go. Script. Alternatively, some thermostable DNA polymerases (e.

Tth DNA polymerase) possess a reverse transcriptase activity, which can be activated by using manganese instead of magnesium as a cofactor (Myers and Gelfand, 1. After this initial reverse transcription step to produce the c.

DNA template, basic PCR is carried out to amplify the target sequence. The quality and purity of the RNA template is crucial to the success of RT- PCR. Total RNA or poly(A)+ RNA can be used as the starting template, but both must be intact and free of contaminating genomic DNA. Specific capture of poly(A)+ RNA will enrich a targeted message so that less of the reverse transcription reaction is needed for subsequent amplification.

The efficiency of the first- strand synthesis reaction, which can be related to the RNA quality, also will significantly affect amplification results. Additional Resources for RT- PCRTechnical Bulletins and Manuals. TB2. 20 Access RT- PCR System Technical Bulletin. TM3. 16 Go. Script. Chem. 2. 83, 1. 68. Because many basic keratin genes are located at 7q. Hirosaki rat using RT- PCR.

Expression of kb. RT- PCR was performed using the Access. Quick. Immun. 7. 1, 3. The resultant construct, named p. GEMTStx. 2, was used as a template in PCR to amplify each region of the gene corresponding to Shiga toxin type 2 subunits A and B.

Each PCR product was digested with Bam. HI and Eco. RI, then ligated into p. CDNA 3. 1+ to create p. Stx. 2. Mice then were immunized with either one or both of these constructs and another construct expressing murine granulocyte- macrophage colony- stimulating factor. Expression of each subunit in mouse tissue was verified by RT- PCR using specific primers and the Access. Quick. Pub. Med Number: 1. Hot- start PCR is a common technique to reduce nonspecific amplification due to assembly of amplification reactions at room temperature.

At these lower temperatures, PCR primers can anneal to template sequences that are not perfectly complementary. Since thermostable DNA polymerases have activity at these low temperatures (although in most cases the activity is less than 2.

This newly synthesized region then acts as a template for primer extension and synthesis of undesired amplification products. However, if the reaction is heated to temperatures > 6. Hot- start PCR also can reduce the amount of primer- dimer synthesized by increasing the stringency of primer annealing.

At lower temperatures, PCR primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer dimer, which often appears as a diffuse band of approximately 5. The formation of nonspecific products and primer- dimer can compete for reagent availability with amplification of the desired product. Thus, hot- start PCR can improve the yield of specific PCR products. To perform manual hot- start PCR, reactions are assembled on ice or at room temperature, but one critical component is omitted until the reaction is heated to 6.