Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

Bacteria isolated from a pentachlorophenol (PCP) contaminated site grew in the presence of 50 µg PCP/mL but were not able to degrade it in either liquid medium or the presence of 1% sterile potting soil as a solid support. Probes developed using the gene sequence of PCP-4-monooxygenase (pcpB) from Sphingomonas chlorophenolica sp.nov hybridized to two separate isolates. Identification based on fatty acid methyl ester profiles (Sherlock), substrate utilization (BIOLOG), and 16S rRNA showed that the two strains were different from each other and from Sphingomonas chlorophenolica. Sequences from these isolates, amplified by polymerase chain reaction, confirmed the homology with pcpB. The presence of pcpB sequences in these nondegraders indicated that growth and hybridization data alone were insufficient for predicting degradation capability. Key words: pentachlorophenol, Sphingomonas chlorophenolica, pcpB gene, pentachlorophenol-4-monooxygenase.

Download Full-text

Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.10.8813086 ◽

1996 ◽

Vol 44 (10) ◽

pp. 1205-1207 ◽

Cited By ~ 18

Author(s):

A Dakhama ◽

V Macek ◽

J C Hogg ◽

R G Hegele

Keyword(s):

Polymerase Chain Reaction ◽

Pcr Amplification ◽

Housekeeping Gene ◽

Chain Reaction ◽

Viral Genomes ◽

Negative Results ◽

Beta Actin ◽

Target Nucleic Acid ◽

Formalin Fixed Paraffin Embedded ◽

Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

Download Full-text

A Self-Contained Portable Polymerase-Chain-Reaction System Integrated with Electromagnetic Mini-Actuators for Bi-Directional Fluid Transport

Journal of Mechanics ◽

10.1017/jmech.2011.38 ◽

2011 ◽

Vol 27 (3) ◽

pp. 357-364

Author(s):

B. T. Chia ◽

S.-A. Yang ◽

M.-Y. Cheng ◽

C.-W. Lin ◽

Y.-J. Yang

Keyword(s):

Polymerase Chain Reaction ◽

Fluid Transport ◽

Point Of Care ◽

Reaction System ◽

Thermal Characteristics ◽

Pcr Amplification ◽

Chain Reaction ◽

Rapid Temperature ◽

Small Footprint ◽

Polymerase Chain

ABSTRACTIn this paper, the development of a portable polymerase chain reaction (PCR) device is presented. Integrating electromagnetic mini-actuators for bi-directional fluid transport, the proposed device, whose dimension is 67mm × 66mm × 25mm, can be fully operated with a 5V DC voltage. The device consists of four major parts: A disposable channel chip in which PCR mixture is manipulated and reacted, a heater chip which generates different temperature zones for PCR reaction, a linear actuator array for pumping PCR mixture, and a circuit module for controlling and driving the system. The advantages of the device include the rapid temperature responses associated with continuous-flow-type PCR devices, as well as the programmable thermal cycling associated with chamber-type PCR devices. The thermal characteristics are measured and discussed. PCR amplification is successfully performed for the 122 bp segment of MCF-7/adr cell line. Due to its small footprint, this self-contained system potentially can be employed for point-of-care (POC) applications.

Download Full-text