Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project (original) (raw)
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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.
Methods in molecular biology (Clifton, N.J.), 2017
This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide p...
Journal of Forensic Sciences, 2003
Two major goals in the development of new DNA typing technology for forensic use have always been: (1) to achieve the highest power of discrimination possible using a valid and reliable method, and (2) to preserve limited evidentiary samples by utilizing the smallest portion possible for analysis. The discovery of the STR markers (1-3) as well as the development of fluorescence detection instruments (4-9) provided forensic scientists with sophisticated means of achieving their original goals. The development of highly discriminatory megaplex STR systems used in a fluorescence-based detection mode significantly increased the capability for analyzing a larger number of samples that have a limited amount of biological material (10-16). This combined approach offered great sensitivity and accuracy as well as facilitated the interpretation of DNA mixtures by providing data, such as peak height and peak area, that can be integrated into formulas and subsequently analyzed to assist in profile interpretation (17-20). Many validation studies have been published that indicate that STR systems, in multiplex formats, provide excellent typing results for samples subjected to a variety of environmental and experimental conditions (15,21-27). Despite this valuable information, newly developed STR multiplex systems must be subjected, by the practicing forensic community, to a thorough examination in order to define their limitations and establish their robustness and reliability under specific circumstances and/or experimental conditions. Only from such studies can scientifically sound interpretation guidelines be derived and a universal consensus for data sharing among different forensic laboratories be obtained. This report presents some of the studies that were conducted during the validation of the AmpFᐍSTR ® Profiler Plus™ STR amplification system using reduced PCR volume conditions (i.e., 25 L). Although the manufacturers of the AmpFᐍSTR ® commercial kits recommend the use of 50 L as the PCR volume (15,28), previous experience gained using STR multiplexes under reduced PCR volume conditions (21,29) and our involvement in the FBI STR Standardization Project suggested the evaluation of the amplification conditions described in this report. Six different categories of Profiler Plus™ profiles were defined following the processing of 275 biological evidence in 25-L PCR volume. Examples for each category are presented along with data collected and used to develop our interpretation guidelines. As a means to improve the odds of obtaining balanced and complete profiles for casework samples showing partial profiles or profiles with a slope, several amplification conditions are presented. In addition, a series of simulated mixtures representing ten different mixture scenarios was prepared to establish the limit of detection of a minor profile using the Profiler Plus™ multiplex system. Two different amounts of total DNA (1 and 2 ng) and different ratios ranging from 1:20 to 20:1 were selected to cover a wide range of potential casework mixtures. Finally, the specificity of the Profiler Plus™ multiplex amplification reaction was also examined under reduced PCR volume conditions using a large spectrum of nonhuman DNAs, varying the amounts of template DNA (2.5 and 50 ng). The results obtained from these studies are described below.
DNA amplification by polymerase chain reaction (PCR)
HAL (Le Centre pour la Communication Scientifique Directe), 1998
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Distributed under a Creative Commons Attribution-NonCommercial 4.0 International License DNA amplification by polymerase chain reaction (PCR)
A simplified procedure for developing multiplex PCRs
Genome Research, 1995
We have developed a simplified method for multiplex PCR based on the use of chimeric primers. Each primer contains a 3' region complementary to sequence-specific recognition sites and a 5' region made up of an unrelated 20-nucleotide sequence. Identical reaction conditions, cycling times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, efficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additional modification of either the reaction components or annealing temperatures is required. The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.
Clinical Chemistry and Laboratory Medicine, 2000
There are several biological sources which could be used for DNA isolation and genetic testing with potential application in the clinical setting [1]. Increasingly, DNA isolated from buccal swabs and, more recently, saliva samples is under investigation for potential use in the clinical setting rather than traditional blood samples due to ease of access, storage, and transport, as well as relative comfort for participants with low cost of acquisition . Reports to date indicate that DNA isolated from saliva may be more reliable for genotyping by PCR and electrophoresis [3-6], whereas buccal DNA from mouth swabs has produced variable genotyping success as low as 23% . However, there is a paucity of information available on the comparison of buccal and/or saliva DNA for chromosomal microarrays, a commonly used tool for genetic testing. In response to Durdiaková et al.
Pre-PCR Processing : Strategies to Generate PCR-Compatible Samples
Molecular Biotechnology, 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.