Methods and reagents: Vectorette, splinkerette, and boomerang DNA amplification Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses some different methods of amplifying DNA other than by using the standard polymerase chain reaction (PCR). For details on how to partake in the newsgroup, see the accompanying box. Netters seeking novel and creative ways to amplify DNA have come across some interesting methods that can be used when it is not possible to design two flanking primers for the amplification of DNA between them, as is the case when the sequence from the end of a yeast artificial chromosome (YAC) is to be determined. One technique, called inverse PCR, allows for the amplification of DNA away from a single binding site instead of between two primers. This is done by digestion of genomic DNA with a restriction enzyme, ligation of the fragments into small circles, and PCR amplification of the region of interest using two primers situated very close to one another, but pointed in opposite directions. [1,2] Another technique, called vectorette PCR, is also useful when the sequence is known for only one primer-binding site, but it differs slightly from inverse PCR because it involves the digestion of target DNA with a restriction enzyme, ligation of synthetic oligonucleotides called vectorettes onto the ends of the DNA fragments, and then PCR amplification of a specific DNA fragment using both an internal primer specific for the target DNA and another primer capable of annealing to the vectorette. Vectorettes are cleverly designed to contain a central mismatching region, so that they are amplified only when they are attached to the ends of a DNA fragment, and only after extension has occurred from the target primer during the first cycle. [3,4] Another method, splinkerette PCR, is actually a modification of the vectorette PCR technique. Because any 5'-overhanging ends of unligated vectorettes will be filled in during the first extension reaction, vectorettes sometimes initiate priming of the nonspecific DNA fragments in the mixture. Instead of a central mismatch, splinkerettes have mismatches engineered within them so that they form looped-back hairpin structures that will decrease end-repair priming and prohibit the polymerase from nonspecifically priming at the vectorette ends. Using splinkerettes therefore avoids the major problem encountered with vectorettes, i.e. the spurious amplification of non-target DNA. Also, since smaller unwanted fragments are more likely to be selectively amplified, splinkerettes are better suited for amplification of larger DNA fragments. [5] Splinkers from down under? ************************** Someone recently alerted netters about a new technique similar to PCR which has sometimes been mistaken as another Aussie invention. Kevin Ahern (ahernk@bcc.orst.edu) at Oregon State University developed a thermocyclic process of amplifying specific DNA sequences, called boomerang DNA amplification (BDA). The name boomerang comes from the way in which the polymerase begins extension from a single primer-binding site and then makes a loop around to the other strand, eventually returning to the original priming site on the DNA. Although DNA fragments produced by BDA could be analyzed on a gel, the technique should not be confused with Gel boomerangs, which are designed by the world-champion boomeranger Michael `Gel' Girvin. Dr Ahern also warns that, although it could be compared to the polymerase chain reaction (PCR), his new technique should not be called `Boomerang PCR', but rather boomerang DNA amplification (BDA) because, owing to its dependence on only a single primer, the technique is fundamentally different from PCR. The main difference between boomeranging and splinkering is that BDA uses the same oligonucleotide for both primings, and that splinkering uses a second primer that recognizes the sequence within the splinker. Theoretically, DNA amplification using BDA is as easy as ABC. The target DNA is first digested with an endonuclease, which creates a sticky end ready for ligation to an oligonucleotide adapter. A universal adapter is designed so that it has self-complementary ends and a noncomplementary midsection. The adapters are annealed together to form a loop or lollipop structure. (Fig. 1a). Since specific amplification is not dependent on the adapters, they can be made once and used for many different experiments. In the example presented here, the target DNA has a 5'-extruding end, but although it would probably be less efficient, this step could be performed as a blunt-ended ligation, which is not possible with vectorettes because they must be ligated onto the ends in the correct orientation. Next (Fig. 1b), a single specific primer is used for polymerase extension around the bend of the adapter. Once the adaptor has been extended over the top, the extension product creates a newly synthesized primer-binding site, which is used in the next set of denaturing, annealing, and polymerization steps. Cycling of temperature in the presence of a thermostable polymerase of your choice (Fig. 1c) leads to the production of a specific product that can then be cloned and sequenced. Although the technique has not been thoroughly researched and there is still some tweaking to be done to optimize it, boomeranging has so far enabled Dr Ahern to amplify specific target DNA fragments by as much as 10^5-fold. One pitfall, however, is that many different cruciform structures might be present in the mixture, and the amplification of these in combination with polymerase slippage and crossover events could result in a number of mixed recombination products. The resulting DNA might be difficult to interpret on a gel, and any of the products could be misconstrued as the true DNA sequence. But this has also been known to happen with `normal' PCR. [6] Another limitation of BDA is that, as with inverse PCR, unless it is performed with blunt-ended adapters, the DNA must have the restriction enzyme site used for creating the ends on which the adapter is ligated. [7] Therefore, adapters with various restriction sites would have to be used in parallel experiments. On the brighter side, BDA is an exciting new twist to the world of PCR and it should be rather simple for others to make their own adapters and begin using the technique. Another interesting note is that since the US patent on PCR is based on the use of a double primer system, it seems that BDA would not be covered by the current patent laws concerning PCR, and might therefore be a loophole for the use of polymerases with higher fidelity than Taq DNA polymerase. (See TIBS 20, 324-325 for a discussion of thermostable polymerase fidelity and the PCR patent controversy.) Although the use of a single primer for amplification of specific DNA is currently not patented in the US, it probably will be shortly. The boomerang technique has yet to be published in the scientific literature. Keep your eyes open though, because Dr Ahern is also working up an in vivo single primer method for selective amplification of sequences, called hula-hoop cloning (HHC), that even a six-year-old would enjoy. On behalf of all the netters on methods-reagnts, I would like to thank Dr Ahern for sharing his new technique with us before publication. References: [1] Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetics 120, 621-623 [2] Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) Nucleic Acids Res. 16, 8186 [3] Arnold, C., and Hodgson, I. J. (1991) PCR Methods Appl. 1, 39-42 [4] Riley, J. et al. (1990) Nucleic Acids Res. 18, 2887-2890 [5] Devon, R. S. , Porteous, D. J., and Brookes, A. J. (1995) Nucleic Acids Res. 23, 1644-1645 [6] Odelberg, S. J. et al. (1995) Nucleic Acids Res. 23, 2049-2057 [7] Eeles, R. A. and Stamps, A. C. (1993) in Polymerase Chain Reaction (PCR) the Technique and its Applications, pp. 65-71, R. G. Landes Co. ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/sep95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Fidelity of DNA polymerases for PCR. Trends in Biochemical Sciences 20(9):372-373. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************