The polymerase chain reaction (PCR) has changed the way we do life sciences, but the impact of this technology has reached far beyond that, into clinical settings and other fields of study such as gene/drug discovery, forensic science, ecology, archaeology, genetics/molecular biology, and anthropology. Craig M Sampson reports.
The true power of polymerase chain reaction PCR is seen in the fact that a specific DNA sequence can be amplified potentially to billions of copies even if you do not know the sequence you are amplifying. Since Kary Mullis' discovery in 1983 of the idea of a simple method for producing virtually unlimited copies of a specific DNA sequence in a test tube, scientists have continued to develop applications for this nucleic acid amplification technique. As well, associated biotechnology companies have strived to develop more efficient and streamlined procedures by which this technology can be used.
Xtrana Inc has developed nucleic acid binding/amplification applications that streamline this technology via the Xtra Amp Nucleic Acid Extraction Kit. This kit provides a rapid method by which DNA or RNA may be extracted from a variety of biological samples and subsequently amplified by standard PCR procedures in the same tube. Xtra Bind is Xtrana's solid phase nucleic acid binding material that has been incorporated into the Xtra Amp kit. This kit has applications for genomic DNA extraction from whole blood, buffy coat, and tissue culture cells as well as chromosomal or plasmid DNA extraction from E. coli, and total RNA extraction from tissue culture cells.
Applications in development include genomic DNA extraction from buccal swabs, mouse tails, and yeast, plus both genomic DNA and RNA extraction from whole tissue and plant cells. The nucleic acid is irreversibly bound to the solid phase matrix and has shown remarkable stability. The Xtra Bind provides innovative applications such as direct interface with PCR or alternative amplification methods, storage or archiving of bound nucleic acid, repeat gene analysis or multiplex genetic loci detection in series, and rapid automation of nucleic acid extracts.
The entire process eliminates any vacuum filtration, centrifugation, transfer, or dilution steps often involved with other extraction methods. The manual hands-on time of the Xtra Amp kit is about three minutes with bound nucleic acid being available for PCR in 3-15 minutes.
The use of a single tube for both nucleic acid extraction and amplification makes this an ideal system for sample tracking by barcoding and sample processing in automated systems. For example, an entire set of 96 samples can be completely extracted with an automated robotic system in less than 15 minutes or manually with a multichannel pipettor in less than 30 minutes. The Xtra Amp tube is a standard PCR tube which has been coated on its inner surface with the Xtra Bind solid phase material to a level of 50 ml.
To extract the sample, add 50-75ml of lysis buffer to the tube and after allowing 1-3 minutes for resin hydration, add 50-75ml of the sample. This is mixed with the lysis buffer and incubated at room temperature for no more than 10 minutes. The resultant lysate is removed and the solid phase material washed with the wash buffer provided in the kit and then discarded. The Xtra Amp tube is available for amplification via PCR or RT-PCR, with amplification being performed from the nucleic acid bound to the Xtra Bind solid phase surface.
RNA extractions from tissue culture cells can be amplified via RT-PCR using as few as 10 cells or as many as 104 cells. With this kit, we recommend an AMV-RT based reverse transcriptase for the amplification. Likewise, the genomic DNA kit for tissue culture cells works with as few as 10 cells or as many as 104 cells, with the E. coli kit showing extractions with 1-75ml of an overnight broth culture.
The whole blood extraction kit is validated for use with 25-75ml of fresh or frozen samples while the buffy coat kit sees optimal results with 10ml samples. The Xtra Amp kit formats are currently available for either manual or automated processing in eight-tube strips or 96-well plates. A 384-well plate format is in development and will be available later this year. Each kit, regardless of its specific application comes complete with Xtra Amp tubes (12 separate eight-tube strips) or a coated 96-well plate, lysis buffer, wash buffer, Amp Enhance solution, and a detailed protocol.
ENQUIRY No 94
Craig M Sampson is with Xtrana Inc, Broomfierld, CO, USA. www.xtrana.com
