DNA Amplification and Detection Made Simple (Relatively) 英文参考文献.docVIP

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DNA Amplification and Detection Made Simple (Relatively) 英文参考文献.doc

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DNA Amplification and Detection Made Simple (Relatively) 英文参考文献

DNA Ampli?cation and Detection Made Simple (Relatively) Mary Ho? | DOI: 10.1371/journal.pbio.0040222 Twenty-three years ago, a man musing about work while driving down a California highway revolutionized molecular biology when he envisioned a technique to make large numbers of copies of a piece of DNA rapidly and accurately. Known as the polymerase chain reaction, or PCR, Kary Mullis’s technique involves separating the double strands of a DNA fragment into single-strand templates by heating it, attaching primers that initiate the copying process, using DNA polymerase to make a copy of each strand from free nucleotides ?oating around in the reaction mixture, detaching the primers, then repeating the cycle using the new and old strands as templates. Since its discovery in 1983, PCR has made possible a number of procedures we now take for granted, such as DNA ?ngerprinting of crime scenes, paternity testing, and DNA-based diagnosis of hereditary and infectious diseases. needed to bind the primer to the strands, so the reaction chamber must repeatedly cycle through hot and cold phases. As a result, the technique can only be performed in laboratories using sophisticated equipment. Now Olaf Piepenburg, Niall Armes, and colleagues have come up with a new approach to DNA ampli?cation that can be carried out at a constant temperature, using only a tiny amount of DNA, without elaborate equipment. Called recombinase polymerase ampli?cation (RPA), the technique opens the door to dramatically extending the application of DNA ampli?cation in ?eldwork and in laboratories where PCR machines are not available. RPA uses ?ve main ingredients: a sample of the DNA to be ampli?ed; a primer–recombinase complex, which initiates the copying process when it attaches to the template; nucleotides from which to form the new strands; a polymerase, which brings them together in the right order; and single-stranded DNA-binding proteins (SSBs), which As valuable as conventional PCR