scholarly journals High Efficiency Restriction Enzyme–Free Linear Amplification-Mediated Polymerase Chain Reaction Approach for Tracking Lentiviral Integration Sites Does Not Abrogate Retrieval Bias

2013 ◽  
Vol 24 (1) ◽  
pp. 38-47 ◽  
Author(s):  
Chuanfeng Wu ◽  
Alexander Jares ◽  
Thomas Winkler ◽  
Jianjun Xie ◽  
Jean-Yves Metais ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Enzyme ◽  
High Efficiency ◽  
Chain Reaction ◽  
Linear Amplification ◽  
Polymerase Chain Reaction Approach ◽  
Integration Sites ◽  
Polymerase Chain ◽  
Lentiviral Integration ◽  
Retrieval Bias

Detecting multiple cystic fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis

1991 ◽  
Vol 37 (5) ◽  
pp. 753-755 ◽  
Author(s):  
K J Friedman ◽  
W E Highsmith ◽  
L M Silverman
Keyword(s):  
Cystic Fibrosis ◽  
Polymerase Chain Reaction ◽  
Restriction Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chain Reaction ◽  
Allele Specific ◽  
Specific Restriction ◽  
Polymerase Chain

Abstract The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

Thermal Analysis of a Micro-Polymerase Chain Reaction Device

2004 ◽  
Author(s):  
Jeff Punch ◽  
Bryan Rodgers ◽  
David Newport ◽  
Mark Davies
Keyword(s):  
Thermal Analysis ◽  
Polymerase Chain Reaction ◽  
High Efficiency ◽  
Closed Form Solution ◽  
Thermal Control ◽  
Rate Of Change ◽  
Temperature Cycling ◽  
Chain Reaction ◽  
Macro Scale ◽  
Polymerase Chain

Micro-scale polymerase chain reaction (micro-PCR) systems offer substantial advantages over macro-scale systems. Smaller sample volumes are required, and faster process times are feasible. Thermal control of micro-PCR systems is a substantial technical challenge, however. The PCR process requires the fluid sample to be cycled through three temperature ranges — typically 90–95°C, 50–65°C and 72–77°C for denaturation, hybridisation and replication respectively. Durations of the three steps are required to be in the ratio of 4:4:9. In this paper, the thermal analysis of a continuous flow micro-PCR device is reported. The objective of the analysis is to optimize the thermal performance of the device for fast amplification cycles with high efficiency - an efficient PCR features rapid heating and cooling between steps, and good temperature uniformity within each step. The device comprises an array of parallel microchannels formed within a polypropylene substrate to carry fluid, with the base of the substrate mounted on an aluminium carrier. Substrate depth is 500 micron, and each channel is 60 micron wide by 40 micron deep. Thermoelectric cells (TECs) are bonded to the carrier, and powered by a thermoelectric controller with feedback from sensors embedded in the carrier. A Pyrex Glass slide is bonded to the substrate to form closed channels. Arrays of film heaters mounted on the slide adjacent to the channel are used to establish the required temperature regions along the channel. By pumping the fluid at a fixed flow rate, temperature cycling of specific period is achieved. Thermal analysis of the substrate is performed using an approximate closed-form solution, in conjunction with Finite Element (FE) and Computational Fluid Dynamics (CFD) simulations. The analysis is used to conduct a parametric study in order to determine the optimum configurations of substrate materials, cooling conditions, heaters and flow rates required to impose specific temperature cycles. The use of thermoelectric cells is shown to increase the rate of change of temperature between the various regions, improving the efficiency and decreasing the cycle time of the PCR process. Cycle times of 6s or less are shown to be feasible, yielding benefits in time saved for multiple amplifications. Finally, the analysis is also used to identify the dimensionless parameters which govern the thermal characteristics of the device, illustrating the importance of the Biot number.

Apolipoprotein E polymorphism determined by restriction enzyme analysis of DNA amplified by polymerase chain reaction: convenient alternative to phenotyping by isoelectric focusing

1990 ◽  
Vol 36 (12) ◽  
pp. 2087-2092 ◽  
Author(s):  
K Kontula ◽  
K Aalto-Setälä ◽  
T Kuusi ◽  
L Hämäläinen ◽  
A C Syvänen
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetic Variation ◽  
Apolipoprotein E ◽  
Isoelectric Focusing ◽  
Restriction Enzyme ◽  
Enzyme Analysis ◽  
Variant Form ◽  
Chain Reaction ◽  
Apo E ◽  
Polymerase Chain

Abstract Three common alleles determine six apolipoprotein E (apo E) phenotypes that are associated with variations in serum cholesterol in the population. This genetic variation results from single nucleotide alterations at two DNA loci encoding the amino acid residues 112 and 158 of apo E. We compared results of apo E phenotyping carried out by isoelectric focusing with those of apo E genotyping accomplished by direct DNA analysis. In the latter, the target DNA was amplified by the polymerase chain reaction (PCR) and subsequently analyzed by digestion with the restriction enzyme Hha I, followed by polyacrylamide gel electrophoresis of the cleavage products. With one exception, these two techniques yielded similar results from all 40 samples tested. In addition, a rare variant form of apo E (phenotype E1) was analyzed separately and incorrectly diagnosed as E2 by the Hha I digestion method; the anticipated mutation in the codon 127 was, however, confirmed by demonstration of a new Taq I restriction site in this variant gene. These data confirm that the common isoforms of apo E usually arise from genetic variation of the codons 112 and 158 and demonstrate the feasibility of the PCR technique in apo E genotyping.

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