PCR: A brief history
The concept of producing multiple copies of a specific DNA molecule by a cycling process using DNA polymerase and oligonucleotide primers was first expounded in 1971 (Kleppe et al. 1971. J. Mol. Biol. 56: 341-361). The practical exploitation of such a process was hindered by the difficulty and cost of producing oligonucleotides, the non-availability of thermostable DNA polymerases and the lack of automated thermocycling instruments.
By the time of the first demonstration of the PCR process in 1985 (Saiki et al. 1985. Science 230: 1350-1354) automated oligonucleotide synthesizers were commonly available. The potential of PCR in a wide range of applications was recognised. It was still necessary to inject fresh thermo-labile polymerase prior to each elongation step and thermal cyclers were still in development.
The key step in realizing the potential of the PCR was the use of a thermostable polymerase which was first described in 1988 (Saiki et al. 1988. Science 239: 487-491). Since the first description of a practical DNA amplification process many refinements have been described and automatic thermal cyclers have become standard laboratory equipment.
PCR is now an essential tool for many biologists and the standard protocols are simple and user friendly. The exponential amplification process provides nanogram quantities of essentially identical DNA molecules starting from a few copies of a target sequence. The amplified material (the PCR amplicon) is available in sufficient quantity to be identified by size analysis, sequencing, amplicon melting or by probe hybridization. It can also be cloned readily or used as a reagent.
from N.A. Saunders in Real-Time PCR: Current Technology and Applications
Bibliography:
By the time of the first demonstration of the PCR process in 1985 (Saiki et al. 1985. Science 230: 1350-1354) automated oligonucleotide synthesizers were commonly available. The potential of PCR in a wide range of applications was recognised. It was still necessary to inject fresh thermo-labile polymerase prior to each elongation step and thermal cyclers were still in development.
The key step in realizing the potential of the PCR was the use of a thermostable polymerase which was first described in 1988 (Saiki et al. 1988. Science 239: 487-491). Since the first description of a practical DNA amplification process many refinements have been described and automatic thermal cyclers have become standard laboratory equipment.
PCR is now an essential tool for many biologists and the standard protocols are simple and user friendly. The exponential amplification process provides nanogram quantities of essentially identical DNA molecules starting from a few copies of a target sequence. The amplified material (the PCR amplicon) is available in sufficient quantity to be identified by size analysis, sequencing, amplicon melting or by probe hybridization. It can also be cloned readily or used as a reagent.
from N.A. Saunders in Real-Time PCR: Current Technology and Applications
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: history
Real-time PCR
The development of instruments that allow real-time monitoring of fluorescence within a PCR reaction vessel led to a significant advance in PCR technology and applications. Many different instruments and fluorescent probe systems have been developed and are currently available.
Real-time PCR assays can be completed rapidly since no manipulations are required post-amplification. Identification of the 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. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations.
Real-time PCR has applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.
Bibliography:
from N.A. Saunders in Real-Time PCR: Current Technology and Applications
Real-time PCR assays can be completed rapidly since no manipulations are required post-amplification. Identification of the 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. Further investigation of the real-time PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations.
Real-time PCR has applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Books
from N.A. Saunders in Real-Time PCR: Current Technology and Applications
Labels: applications, assays, NASBA, probes, real-time pcr
PCR in Diagnosis: Malaria
It is clearly desirable to develop rapid and precise diagnostic PCR procedures for use in clinical laboratories. The authors of a recent review on the use of PCR for the diagnosis of malaria assert that PCR "should be considered as the gold standard for the diagnosis of imported malaria". Since the first description of the diagnosis of Plasmodium infection by PCR, the role of PCR in the laboratory diagnosis of imported malaria is still a topical question. However, the authors claim that PCR-based assays are more sensitive and more specific than all conventional methods. The highest contribution of the molecular diagnosis is that a negative PCR result would indicate the lack of any malaria infection, thus quickly orienting the investigations toward other aetiology.
from Berry et al. in Parasite 2008 15(3): 484-488
Bibliography:
from Berry et al. in Parasite 2008 15(3): 484-488
Bibliography:
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- Real-Time PCR: Current Technology and Applications
- PCR Books
- Malaria Parasites: Genomes and Molecular Biology
Labels: applications, clinical, diagnosis, microbiology
Reamplification of a PCR Band
The products of a polymerase chain reaction commonly contain a mixture of products due to the amplification of unwanted or non-specific PCR fragments. This results in a range of bands on a gel often referred to as a "ladder" or "smear". Only one of these bands represents the desired DNA fragment. It can be difficult to find the correct PCR parameters with which to obtain the correct band in a pure state while still maintaining yield. Attempting to purify the band by cloning all the reaction products and then probing the library for the correct DNA can be very tedious and time-consuming.
A simple "core sampling" procedure can be used to isolate the required fragment from a mixture of PCR products. This post-PCR procedure involves "coring" an agarose sample out of a gel and using the sample as template in another round of PCR. This is a good "work-around" to obtain unique bands from a background of a large number of unwanted DNA fragments. Having a visible band of the required size is recommended although it is still possible to attempt the technique on "right-sized" invisible bands if a visible band cannot be achieved.
The full protocol for this PCR technique is available on-line at Core-Sampling a PCR Band
Bibliography:
A simple "core sampling" procedure can be used to isolate the required fragment from a mixture of PCR products. This post-PCR procedure involves "coring" an agarose sample out of a gel and using the sample as template in another round of PCR. This is a good "work-around" to obtain unique bands from a background of a large number of unwanted DNA fragments. Having a visible band of the required size is recommended although it is still possible to attempt the technique on "right-sized" invisible bands if a visible band cannot be achieved.
The full protocol for this PCR technique is available on-line at Core-Sampling a PCR Band
Bibliography:
- PCR Troubleshooting: The Essential Guide
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Books
Labels: core sampling, PCR cloning, protocols, reamplification
The PCR Blog
The polymerase chain reaction (PCR) is a laboratory technique that amplifies a specific DNA fragment. PCR is used to amplify DNA molecules for subsequent laboratory manipulation (for example cloning or DNA sequence analysis) and also for sensitive detection tests (for example in clinical diagnostics). The advantages of PCR over other methods include rapidity, specificity and sensitivity.
The PCR technique is extremely useful in basic research, for commercial uses, genetic testing, forensics, industrial quality control, environmental science, food microbiology and clinical diagnostics. PCR is now a very commonplace procedure in all molecular biology laboratories where it is used to amplify DNA fragments and detect DNA or RNA sequences. Many variations and improvements of the original PCR method have been developed.
Despite being a relatively simple procedure, PCR can be infuriatingly problematic. Depending on the nature of the DNA fragment being amplified and the biological properties of the primers, the PCR procedure can fail totally, produce insufficient DNA product, or (commonly) amplify unwanted, non-specific fragments. In order to optimize a PCR procedure it is necessary to consider a huge range of variations and permutations of the original procedure.
This blog provides a current and regularly updated resource on different types of PCR technology, methods, applications and PCR optimization. In addition, we provide regular features on PCR trouble shooting. Where a detailed treatment of the topic is beyond the scope of the blog we provide a bibliography for scientists and researchers who require more comprehensive information.
To contribute to this blog please contact us
The PCR technique is extremely useful in basic research, for commercial uses, genetic testing, forensics, industrial quality control, environmental science, food microbiology and clinical diagnostics. PCR is now a very commonplace procedure in all molecular biology laboratories where it is used to amplify DNA fragments and detect DNA or RNA sequences. Many variations and improvements of the original PCR method have been developed.
Despite being a relatively simple procedure, PCR can be infuriatingly problematic. Depending on the nature of the DNA fragment being amplified and the biological properties of the primers, the PCR procedure can fail totally, produce insufficient DNA product, or (commonly) amplify unwanted, non-specific fragments. In order to optimize a PCR procedure it is necessary to consider a huge range of variations and permutations of the original procedure.
This blog provides a current and regularly updated resource on different types of PCR technology, methods, applications and PCR optimization. In addition, we provide regular features on PCR trouble shooting. Where a detailed treatment of the topic is beyond the scope of the blog we provide a bibliography for scientists and researchers who require more comprehensive information.
To contribute to this blog please contact us
| Social Bookmarking: | Help! What is this? |
|
|
The text of this web page may be used under the GFDL license
