DNA World

Real-Time PCR Papers

An Introduction to Real-Time PCRN.A. SaundersThe development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance. The technology is very flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed very 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.
Real-Time PCR PlatformsM.J. Logan and K.J. EdwardsReal-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software.
Homogeneous Fluorescent Chemistries for Real-Time PCRM.A. Lee, D.J. Squirrell, D.L. Leslie, and T. BrownThe development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes.
Performing Real-Time PCRK.J. EdwardsOptimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence.
Internal and External Controls for Reagent ValidationM.A. Lee, D.L. Leslie and D.J. SquirrellPCR applications that require a high confidence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls.
Quantitative Real-Time PCRN.A. SaundersUnlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real-time PCR improves its performance and a range of suitable systems is now available.
Analysis of mRNA Expression by Real-Time PCRS.A. Bustin and T. NolanThe last few years have seen the transformation of the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) from an experimental tool into a mainstream scientific technology. Assays are simple to perform, capable of high throughput, and combine high sensitivity with exquisite specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology.
Mutation Detection by Real-Time PCRK.J. Edwards and J.M.J LoganReal-time PCR is ideally suited for analysis of single nucleotide polymorphisms (SNPs) and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements.
The Quantitative Amplification Refractory Mutation SystemP. Punia and N.A. SaundersThe amplification refractory mutation system (ARMS), which has also been described as allele-specific PCR (ASP) and PCR amplification of specific alleles (PASA), is a PCR-based method of detecting single base mutations. ARMS has been applied successfully to the analysis of a wide range of polymorphisms, germ-line mutations and somatic mutations. The technique has the ability to discriminate low-levels of the mutant sequence in a high background of wild-type DNA. In an ARMS PCR the terminal 3' nucleotide of one of the PCR primers coincides with the target mutation. Most applications of the method rely on 'end-point' analysis, utilising the classic gel-electrophoresis method.
Real-Time NASBAS. Hibbitts and J.D. FoxNASBA is an isothermal nucleic acid amplification method that is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The first developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and analysis of RNA (and in one case DNA) targets.
Applications of Real-Time PCR in Clinical MicrobiologyA.D. SailsThe introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents. However, its greatest impact is probably its use for the quantitation of target organisms in samples.
Application of Real-Time PCR to the Diagnosis of Invasive Fungal InfectionN. Isik and N.A. SaundersThe management of invasive fungal infections has been hampered by the inability to make a diagnosis at an early stage of the disease. Molecular diagnosis by PCR appears very promising since fungal DNA can be detected in the blood of infected patients earlier than when using conventional methods. Recently, interest in the diagnosis of invasive fungal infections by real-time PCR has increased. Real-time methods also have quantitative properties and are useful both for initial diagnosis and to assess the response to treatment. Many recent studies have combined serological tests with measurement of fungal DNA by using real-time PCR. Real-time PCR helps early diagnosis and arrangement of treatment protocols for patients with high risk of fungal infection.
General PCR Articles
Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping Curr. Issues Mol. Biol. (1999) 1: 77-88 Chengtao Her and Richard M. WeinshilboumThe polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR.
A PCR-based Method for Isolation of Genomic DNA Flanking a Known DNA Sequence Curr. Issues Mol. Biol. (1999) 1: 47-52 Catherine A. Boulter and Dipa NatarajanA simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence.
Universal TA Cloning Curr. Issues Mol. Biol. (2000) 2: 1-7 Ming-Yi Zhou and Celso E. Gomez-SanchezTA cloning is one of the simplest and most efficient methods for the cloning of PCR products.
Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction Curr. Issues Mol. Biol. (2002) 4: 13-18 Clifford F. Brunk, Jinliang Li and Erik Avaniss-AghajaniThe use of quantitative PCR for measuring bacterial abundance in environmental samples.
PCR Clamping Curr. Issues Mol. Biol. (2000) 2: 27-30 Henrik ØrumAn efficient, PCR based method for the selective amplification of DNA target sequences that differ by a single base pair. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
DNA Splicing by Directed Ligation (SDL) Curr. Issues Mol. Biol. (1999) 1: 21-30 DNA Yuri A. BerlinSplicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites.
_PCR Family Brochure.book
File Format: PDF/Adobe AcrobatA basic set of PCR (chapter 4) and RT-PCR (chapter 5) protocols, including tips on. how to get the best results with our products. ...www.roche-applied-science.com/PROD_INF/MANUALS/pcr_man/chapter_1.pdf

PCR--Polymerase Chain Reaction

PCR—from (Dr. Chen, Dept of Biochem. & Mol. Biology, Univ. College London)

Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers

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