T4 contains a 168 kb dsDNA genome and adds a known amount of DNA, when added to a sample.
Primers, probe, and master mix are available for amplification and detection of T4 DNA. The T4 primers are specific for a 163 bp portion of the tail tube glycoprotein 18 gene. The T4 detection probe is a molecular beacon.
Bacteriophage T4 was likely isolated by Demerec and Fano (1945) from a phage mixture provided by Dr. Tony L Rakieten,(1) The Long Island College of Medicine. The original source is speculated to be from a lysate inoculated with raw sewage.
Extraction / amplification control with T4 bacteriophage
- Used as an Internal control (Multiplex reaction)
- Adding T4 bacteriophage (BAC130) to the sample provides DNA for extraction, amplification, and reaction condition PCR controls. When added to a sample, T4 adds a known amount of DNA. The T4 DNA can then be extracted, amplified, and detected as a control. T4 controls for the efficiency of DNA extraction, the presence of PCR amplification inhibitors, intact amplification reagents ( DNA polymerase, buffer, dNTPs), and instrument functions (thermal cycling and fluorescence detection). The T4 DNA may be detected using a multiplex reaction using Quasar 670 labeled T4 probe (PP160) and FAM labeled test organism probe.
- Used as an External Control
- Extract T4 DNA. Then in a separate reaction vial add Master Mix and extracted DNA. This T4 method controls for the efficiency of DNA extraction, the presence of PCR amplification inhibitors, intact amplification reagents ( DNA polymerase, buffer, dNTPs), and instrument functions (thermal cycling and fluorescence detection). The T4 DNA may be detected in a separate PCR reaction using FAM labeled T4 probe (PP100) in your master mix or Attostar’s MM150 [Quasar 670] or MM160 [FAM].
Brief procedure for use of T4 as extraction and amplification control:
- Add 5µl bacteriophage to the sample. Proceed with DNA extraction.
- Dilutions of the bacteriophage may be made to give a final PCR Ct value that is about 35. At this dilution, the phage is more sensitive, (i.e. more likely, to detect a poor extraction or the presence of PCR inhibitors in the reaction). If multiplex reactions are being used, this dilution is less likely to compete with the test organism PCR reaction.
Use of the T4 plasmid:
- Dilute the plasmid in TE to prepare a standard curve. Common dilutions would be 10-fold from 200 to 0.02pg/ml. The 0.02 pg/ml plasmid dilution contains 12 copies of plasmid in 2 µl.
DNA-PCR and RNA-RT-PCR assays need to control for the following:
- Extraction of DNA or RNA.
- DNA-PCR and RNA RT inhibition.
- Reagent quality including dNTPs, Buffers, Taq, and reverse transcriptase.
- Instrument function, including thermal cycling and fluorescent detection.
The Attostar answer to these control questions is to use bacteriophage–T4 for DNA and Qß for RNA. The Attostar regents include:
- Extraction Control,
- T4 and Qß bacteriophage,
- PCR master mixes,
- Primers and probe reagents,
- Plasmid DNA.
Detection of the bacteriophage internal control DNA/RNA signifies a successful extraction and amplification.
Failure to detect the bacteriophage internal control DNA/RNA means the extraction or amplification procedure failed.
To use these reagents, T4 or Qß is added directly to the sample. The sample is then extracted for DNA or RNA. The extracted nucleic acid, now containing T4 or Qß and the sample nucleic acids are used for PCR or RT-PCR.
The probe for real time detection of this T4 DNA or Qß RNA target is a fluorophore such as FAM or Quasar 670 and a quencher.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
1: This product is sold under license from the Public Health Research Institute. It may be used under PHRI Patent Rights only for the purchaser’s research and development activities.
2: ‘Black Hole Quencher,’, ‘CAL Fluor,’ ‘Pulsar’ and ‘Quasar ’ are trademarks of and licensed by Biosearch Technologies, Inc., Novato, CA. The BHQ, CAL Fluor, Pulsar and Quasar dye technology is the subject of existing or pending patents including US Patent No. 7,019,129 and is licensed and sold under agreement with Biosearch Technologies, Inc.