Methods and reagents

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email: sokoMkc.denison.edu. Methods and ... startiqj template was of such low quart- tlty and the band of ... An even easier approach is to stick a sterile toothpick ...
publication. electronic Pr&in-2-5.txt, and C~Kin.brt describe in varying detail the use of the two programs. Demo3Jakin and Demo3Jbkin are files that contain excellent demon&rations of what can be created with ~~~by~~~~r, the most important files available from the elecmnk lw&l Scienceare those that contain the programs themselves. It is not often that such a powerful

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2 Richardson, 0.6. and Richardson, Sci. 1,3-9 2 Richardson, 0. C. and Richardson,

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J. S. (19921 J. S.

(1994)TrendsBiochem. sci. 19, 135-138 3 Kafplus, P. A., Daniets, M. 1. and Herriott,

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email: sokoMkc.denison.edu

although different concentrations in the secondary PCR also gave a different type of smear In addition, changing the annealing temperature, the amount of poiymerase or the number of cycles in either the primary or the secondary Reamplfficationof PCRfragments PCRdid not correct the problem. column that highlights currant discussions in Unfortunately, this poor netter susavahble on the Internet. This month’s column pected that the problem was a combiwhile trying to reamplify DNA made by nation of all the various parameters dispertake in the nawsgroup, see the accussed, and he continued to experience box the smears. Someone su Andrew lbiymke (zool2Ol&sc.canter- template for reamplification of the try ‘touchdown’ PCR, a method by desired DNA fragment - smears were which the annealing temperature is .nz)wastryingtoampllfya1kb ofmltochondrialDNAfroma seen on an agarose gel every time. In incrementally lowered during each human sperm cell using primers ‘Frohmanlan’ terms, this common result cycle of the PCR, and that this might speclflc to the target gene. Since the is often referred to as ‘ampii-schmutz’i help to determine the anneaiing temstartiqj template was of such low quart- and is mostly caused by nonspecific perature at which a more specific pritlty and the band of interest was nearly amplification in the original PCR. mary product can be generated3”. erthe und of the It is also likely that the smears are Someone else suggested that chop chain by using far too much DNA in ping up the smear with restriction (?pcQ* he ~~~~~~ PCR Reampllfication enzytxs known not to cleave within the iLz?m to inem the yield of the works well when the concentration of PCR product of Merest might help altemplate DNA ls quite low. Therefore, leviate the smears. Another way would difndm. the primary PCR product must be di- be to design nested primers, which are luted at least 10 OOO=fold before reampli- complementary to sequences that lie fkath. However, even when the prl- within the fragment generated by the mazy mixture of his DNA was diluted first round of amplifications. down to as We as 0.1 pg of template If a DNA band of the expected size DNA&3mearwasnotresolvedlntoa can be seen on a gel as well as a backdiscreteband. ground of smeared DNA, an effective Short of suggesting that he should method used to improve reamplifidesign a new set of ollgonucleotide cation yield and eliminate most of the primers more specific for the target unwanted products is gel purification ar with the problem or size fractionation of the band of might improve the interest. Since slicing a small piece of polythe inkiai ~~~ condlti. For acrylamide gel is very difficult to do, -pkthtsmrvybedonebyLnneasone person suggested that he extract ter+xature, or by the DNA from the gel by cutting a * concentration, the trough just ahead of the m&rating DNA number of cycles, the amount of en- band and filling it with low-meltingzyme, or the concentration of dNTPs2. point agarose. Electrophoreslng the gel WMly the h&f+ concentration was horizontally at approximately 10 V cm-* opthkd for the primary PCR, for MOmin in a submarine-style

Methods andreagents

apparatus would cause the DNA to migrate into the agarose. The gel slice could then be melted at 65°C and diluted with Tris-EWl‘A buffer to recover the DNA. After the amount of DNA has been estimated, I-tong can be added to a pre-assembled PCR reaction lacking template for the next round of PCR Of course, once the DNA has been electro-eluted from polyacrylamide into agarose, any of the methods to recover DNA fragments from agarose gels can be used, as was discussed previously in Methods and mqfents [7IaS 19 (1994). 388-3891. Alternatively, the remaining DNA from the primary reaction mixture could be loaded directly onto an agarose gel and the band sliced out. Unfortunately, a low concentration of PCR product would most probably be invisible, even with the best detection dye available. If the band could be seen and extracted, the DNA could be diluted with 1 ml of water and boiled for 5 min before adding 1-5 pl to the other components for the secondary PCR An even easier approach is to stick a sterile toothpick or needle into the gel at the site of the banded fragment, and to swish this through a new PCR reaction mix in order to inoculate it with a tiny amount of specific DNA6s7. An elegant trick is to have one of the PCRprimers biotinylated. 10 pl of Dynal M-290 streptavidin-coated pararnagnetic particles (Dynal, Inc., Great Neck, KY,USA)are washed in 500 ~1of binding buffer (50 mM Tris-HCI, 1OmM EDT%, 1 M NaCI, pH 7.6). The solution is loaded into a small syringe fitted with an l&g?uge needle or smaller. A single PCR+mplified hand resolved on a polyacrylamide gel can be recovered by stabbing the needle into the band and pumping the buffer and beads gently in and out. If the gel is stained with SYBR Green I (A4olecular Probes, Inc., Eugene, OR, USA)and placed on top of a 254 nm transillurnlnator, the green dye can be seen moving up into the syringe. Afterwards, the beads are recovered with strong magnets, washed twice with 2004 of Tris-EDTA, and resuspended in 10 p1 of sterile water. Then 1 pi of the beads is introduced into a fresh PCR

mix to reamplify the fragment. After RX, the beads can be recovered by magnets or centrifugation and the template can be reused. In addition, the attached DIVA template can be used for sequencing directly by solid-phase cycle sequencing! Although Mr Holyoake did not obtain the desired result, he was encouraged by a more positive outcome - another netter experiencing the same problem found a solution. After having difficulty pinpointing a single cause for the smears, careful cansideration of all the materials used allowed this netter to determine that one of the contributing factors was an excess of one primer, which may have interfered with the amplification. R

1 Fmhman, M. A. (1994) in i7ie Wyfrwase cItein(hMb, K. B., Fen& F. artd Gibbs, R. A., eds) p. M-37. Birkehiiuser

ZCatinari, M. etal. (1993) TrendsGmet. 9, 42-43 3 Don, R. H. etaf.(lQQl) /U~~Res. 19, 4 ii.tf K. Ii. (1994) Biot"W16, 812-814 5 Damiger, lx s., !iizF?i;tg

l3ioi&*14,370 6 Kadobmi. Y. and lmiis, R. V. (1994) 8iorechnklugs 17.438 7Bjoumon.A.J.and Coopar, 1. E. (1992)

!vucleicAcids R%s.20, 4675

8 HuItman, T. et al. (1989)

NuckicAckfsRes.17, 4937-4946

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