Pre-PCR Processing : Strategies to Generate PCR-Compatible Samples (original) (raw)

Strategies to Generate PCR-Compatible Samples

2004

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCRinhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCRamplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

PCR primer: a laboratory manual

2003

From its first-published account in 1985, the polymerase chain reaction has become a standard research tool in a wide range of laboratories. Its impact has been felt in basic molecular biological research, clinical research, forensics, evolutioer~y studies, and the Human Genome Project. The PCR technique originally conceived by Nobel laureate Kary Mullis has proven to be exceptionally adaptable and has been transformed into a myriad array of methods, each with different applications. PCR Primer: A Laboratory Manual introduces the complex world of PCR by beginning at an accessible level and then moving to more advanced levels of application. First, the practical requirements for performing PCR and other amplification techniques in the lab are introduced and then the basic aspects of the technique are explained by exploring important issues such as sample preparation, primer design, efficiency, detection of products, and quantitation. Protocols for a wide range of PCR and amplification techniques-each written by an expert investigator.are presented for cloning, sequencing, mutagenesis, library construction and screening, exon trapping, differential display, and expression, and these include RT-PCR, R N A PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3 ' and 5 ' RACE, in situ PCR, and iigationmediated PCR. Each protocol is augmented by analysis and troubleshooting sections and complete references.

Polymerase Chain Reaction (PCR): Back to Basics

Advanced molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production, health, environment, industry other related areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. PCR is a process used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Mechanisms involved in this methodology are similar to those occurring in vivo during DNA replication. Through this paper we will review procedure, advantages, types & applications of PCR.

Specificity, efficiency, and fidelity of PCR

Genome Research, 1993

The efficacy of PCR is measured by its specificity, efficiency (i.e. yield), and fidelity. A highly specific PCR will generate one and only one amplification product that is the intended target sequence. More efficient amplification will generate more products with fewer cycles. A highly accurate (i.e., high-fidelity) PCR, will contain a negligible amount of DNA polymerase-induced errors in its product. An ideal PCR would be the one with high specificity, yield, and fidelity. Studies indicate that each of these three parameters is influenced by numerous components of PCR, including the buffer conditions, the PCR cycling regime (i.e., temperature and duration of each step), and DNA polymerases. Unfortunately, adjusting conditions for maximum specificity may not be compatible with high yield; likewise, optimizing for the fidelity of PCR may result in reduced efficiency. Thus, when setting up a PCR, one should know which of the three parameters is the most important for its intended application and optimize PCR accordingly. For instance, for direct sequencing analysis of a homogenous population of ceils (either by sequencing or by RFLP), the yield and specificity of PCR is more important than the fidelity. On the other hand, for studies of individual DNA molecules, or rare mutants in a heterogeneous population, fidelity of PCR is vital. The purpose of current communication is to focus on the essential components of setting up an effective PCR, and discuss how each of these component may influence the specificity, efficiency, and fidelity of PCR. SETTING UP PCR Template Virtually all forms of DNA and RNA are suitable substrates for PCR. These include genomic (both eukaryotic and prokaryotic), plasmid, and phage DNA and previously amplified DNA, cDNA, and mRNA. Samples prepared via standard molecular methodologies (1) are sufficiently pure for PCR, and usually no extra purification steps are required. Shearing of genomic DNA during DNA extraction does not affect the efficiency of PCR (at least for the fragments that are less than-2 kb). In some cases, rare restriction enzyme digestion of genomic DNA before PCR is suggested to increase the yield. (2'3) In general, the efficiency of PCR is greater for smaller size template DNA (i.e., previously amplified fragment, plasmid, or phage DNA), than high molecular (i.e., undigested eukaryotic genomic) DNA. Typically, 0.1-1 pLg of mammalian genomic DNA is utilized per P e R. (1'3'4-6) Assuming that a haploid mammalian genome (3x109 bp) weighs-3. 4 x 10-az grams, 1 ~g of genomic DNA corresponds to-3 x 10 s copies of autosomal genes. For bacterial genomic DNA or a plasmid DNA that represent much less complex genome, picogram (10-12 grams) to nanogram (10-9 grams) quantities are used per reaction. (1'3) Previously amplified DNA fragments have also been utilized as PCR templates. In general, gel purification of the amplified fragment is recommended before the second round of PCR. Purification of the amplified product is highly recommended if the initial PCR generated a number of unspecific bands or if a different set of primers (i.e., internal primers) is to be utilized for the subsequent PCR. On the other hand, if the amplification reaction contains only the intended target product, and the purpose of the subsequent PCR is simply to increase the overall yield utilizing the same set of primers, no further purification is required. One could simply take out a small aliquot of the original PCR mixture and subject it to a second round of PCR. In addition to the purified form of DNA, PCR from cells has also been demonstrated. In this laboratory, direct amplification of hprt exon 3 fragment from 1 • 10 s human cells (following proteinase treatment to open up the cells) had been carried out routinely (P. Keohavong, unpubl.).

Effects of Amplification Facilitators on Diagnostic PCR in the Presence of Blood, Feces, and Meat

2000

The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.

Polymer Chain Reaction (PCR): Principle and Applications

Ibn AL- Haitham Journal For Pure and Applied Sciences, 2021

The new, standard molecular biologic system for duplicating DNA enzymatically devoid of employing a living organism, like E. coli or yeast, represents polymerases chain reaction (PCR). This technology allows an exponential intensification of a minor quantity of DNA molecule several times. Analysis can be straightforward with more DNA available. A thermal heat cycler performs a polymerization chain reaction that involves repeated cycles of heating and cooling the reactant tubes at the desired temperature for each reaction step. A heated deck is positioned on the upper reaction tube to avoid evaporating the reaction mixture (normally volumes range from 15 to 100 l per tube), or an oil layer can be placed on a reaction mixture surface. The amplified DNA fragment is determined based on selecting primers in addition to the starting and end of the DNA fragment. The primers stand for short, artificial DNA stripes, no higher than fifty (typically 18-25bp) nucleotides have been based on a st...

Polymerase Chain Reaction (PCR): A Short Review

Anwer Khan Modern Medical College Journal, 2013

Diagnosis of disease now a days is mostly laboratory dependent. Due to recent advances in medical science and molecular biology, most of the diagnosis of uncommon, complicated, unusual presentation of disease has left the option of molecular diagnosis as the number one diagnostic modalities. Many molecular techniques are now being widely used throughout the world including PCR, flow cytometry, tissue microarray, different blots, and genetic diagnosis. Among these PCR is the most widely accepted, commonly used diagnostic modalities with very high specificity and sensitivity for correct diagnosis. We have reviewed the principle, application, advantages and disadvantages of PCR in laboratory diagnosis of disease. DOI: http://dx.doi.org/10.3329/akmmcj.v4i1.13682 AKMMC J 2013: 4(1): 30-36

PCR as a specific, sensitive and simple method suitable for diagnostics

Biochemistry and Molecular Biology Education, 2000

PCR technology is a widespread method that has not reached students laboratory in anything else than a typical ampli"cation reaction. We describe a simple application of PCR in pathogen diagnostics that enables students to identify which ampicillin-resistant organism is present in a cell culture. This experiment has been performed for one year in two`Experimental Biochemistry and Molecular Biologya courses with Biological and Chemical undergraduates. Using speci"c primers from the Escherichia coli-lactamase gene, they have been able to selectively amplify a-lactamase DNA fragment in E. coli but not in Staphylococcus aureus and, using di!erent annealing temperatures, test the reaction speci"city. By solving the`Study Questionsa, students understood the speci"city and sensitivity of the method, as well as the rationale that should be applied when a molecular weight pattern is used for calculating unknown DNA sizes.