Methods and reagents: Optimizing multiplex and LA-PCR with betaine

by Paul N. Hengen, Ph.D. *

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 the use of additives for optimizing the amount and quality of product obtained through multiplex and `long and accurate' polymerase chain reaction (LA-PCR). For details on how to partake in the newsgroup, see the accompanying box.

The technique of amplifying long stretches of DNA by the polymerase chain reaction (LA-PCR) has grown in popularity (see TiBS 19, 341-342), and the use of thermostable DNA polymerase mixtures for PCR and cycle sequencing is now commonplace. When amplifying DNA for quantitative PCR, multiplex PCR (amplification of more than one DNA fragment per reaction) or for diagnostic screening purposes using PCR, control primers to sequences known to reside within the template should be included for comparison to the amplicon being tested.

BAFLing results
One problem noted recently in the methods newsgroup is that, even though the sequence being screened for might be present in the template DNA, false negatives can occur so that only the control DNA band appears on a gel. Taq DNA polymerase can stall within particularly difficult regions of template DNA during extension owing to the formation of secondary structures. This can cause low efficiency amplifications in the PCR, or under conditions designed for multiplex amplifications, the expected target DNA can be outcompeted by the more-efficient amplification of control sequence. Stalling can also occur during cycle sequencing reactions, which might result in banding artifacts such as bands in all four lanes (BAFLs) on a denaturing polyacrylamide gel.

False negatives, `band drop-outs' or BAFLs can sometimes be overcome by the elimination of buffer components that stabilize odd secondary structures, such as KCl, or by the addition of co-solvents such as DMSO and glycerol to the PCR mix (see TiBS 21, 33-34). In attempts to overcome these kinds of problems, however, netters sometimes try many different enzymes, co-solvents, and buffer conditions without much success. They complain that some difficult DNAs just will not amplify even when using some of the commercial kits designed for high GC-content PCR. For example, one netter wrote that after trying both the Advantage-GC PCR kit from Clontech and the Q-solution supplied with Qiagen's PCR kit, he still could not get a difficult region to amplify. Also, addition of tetramethylammonium chloride (TMAC) as suggested by others [1,2] did not help.

Betaine
Recently, someone questioned the use of a relatively new additive that can increase the amplification products from high-GC-containing sequences in multiplex PCR. On the information sheet for LA-PCR provided by Wayne Barnes (wayne@barnes1.wustl.edu) available from http://mbb.wustl.edu/~barnes/faq.wpa, it is now recommended that 1.3 M betaine (N,N,N-trimethylglycine; Sigma no. B-2629) and 1.3% DMSO be added to LA-PCR mixtures to improve processivity [3].

Dr Barnes suggests that betaine be added to LA-PCR, that the melting step of each cycle should be reduced to 92-93 degrees C, and the annealing temperature within the PCR cycles should be reduced by 1 or 2 degrees C to compensate for the change in annealing conditions and some decreased enzyme stability caused by the additive.

He wrote that, not only are high-GC-containing targets more easily amplified by including betaine, but that improvements are also seen in the amount of product when it is added to the ordinary type of PCR, i.e. amplifying ~5 kb of DNA. In addition, when used in cycle-sequencing reactions on some more difficult targets, BAFLs can be eliminated.

Another advantage of using betaine is that it acts as an osmoprotectant, and much like BSA, it increases the resistance of the polymerase to denaturation. Replacing BSA with betaine also could help reduce the smearing problem thought to be caused by BSA within LA-PCR buffers. Betaine also allows the PCR to overcome some low level of contaminants that can co-purify with DNA, allowing PCR with DNA samples of lesser quality [4].

As one might expect, the use of betaine does have its drawbacks. Exactly what it is doing to aid in the processivity of Taq is not known. In a recent study [5] on the use of 2 M betaine within sequencing reactions performed with double-stranded supercoiled plasmid DNA templates and T7 DNA polymerase (SequenaseTM), it was shown that the inability to bypass secondary structures can be chased by adding betaine even after a pause has occurred. This suggests that betaine alleviates the paused extension of primer, rather than affecting either the initial annealing of primer to template or the half-life of polymerase, and that betaine somehow disrupts the contorted DNA helix without perturbing the polymerase-DNA interaction.

It has been suggested that betaine affects the extension reaction either by binding to AT pairs in the major groove [5], or by increasing the hydration of GC pairs by binding within the minor groove and thus destabilizing GC-rich DNA [6]. In any case, it appears that betaine is helping in a way different from stabilizing the enzyme. What is of concern is that it is unknown what effect all this has on the fidelity of Taq polymerase, as no data are yet available comparing misincorporation rates of Taq with and without betaine (see TiBS 20, 324-325 for a discussion on the dangers of PCR sequencing). In addition, changes in the amplification efficiencies of various targets owing to betaine could make quantitation of end products more difficult to interpret.

Interestingly, TMANO (trimethylamine N-oxide) seems to work equally as well in preventing polymerase stalls as does betaine. However, TMANO is about ten-fold more expensive. Unfortunately, it appears that comparing the cost of betaine and TMANO is not the major issue facing researchers wanting to use the additives in the PCR. More disturbing perhaps is a patent covering the use of betaine in any DNA or RNA polymerase buffer issued to a German research group (German Patent No. P 44 11 588 1-41).

Burning the midnight oil
Having troubles with your PCR reactions, even after working so hard to rid yourself of contaminants with a UV lamp? Well, it just might be your bottle of oil overlay. Recently, one netter was having difficulties with what should have been a routine amplification of known DNA sequence. A PCR experiment that previously worked well suddenly gave no product band when viewed on an ethidium bromide-stained agarose gel. After testing the separate components of the PCR mix, this person traced the problem to a bottle of mineral oil that he had left sitting under the UV light for a month.

Netters were quite familiar with this problem and could quickly offer a solution. According to one study, mineral oil goes off under 254 nm UV light and the breakdown products can inhibit the PCR, presumably owing to oxidation [7]. Netters say that small volumes of oil (25-30 ml) should be irradiated only briefly and that adding 0.1% of the antioxidan 8-hydroxyquinoline before UV treatment can increase the life of your mineral oil stock, and it could actually help increase the yield of PCR product [8]. They also recommend that mineral oil not be stored under the UV lamp for extended periods of time.

References

[1] Hung, T., Mak, K. and Fong, K. (1990) Nucleic Acids Res. 18, 4953

[2] Chevet, E., Lemaitre, G. and Katinka, M. D. (1995) Nucleic Acids Res. 23, 3343-3344

[3] Baskaran, N. et al. (1996) Genome Res. 6, 633-638

[4] Weissensteiner, T. and Lanchbury, J. S. (1996) BioTechniques 21, 1102-1108

[5] Rees, W. A. et al. (1993) Biochemistry 32, 137-144

[6] Mytelka, D. S. and Chamberlin, M. J. (1996) Nucleic Acids Res. 24, 2774-2781

[7] Dohner, D. E., Dehner, M. S. and Gelb, L. D. (1995) BioTechniques 18, 964-967

[8] Gilgen, M. et al. (1995) Nucleic Acids Res. 23, 4001-4002

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/jun97.txt

Any reference to this column must be cited as the following published article:

@article{Hengen1997Juntibs,
author = "P. N. Hengen",
title = "Methods and reagents - Optimizing multiplex and {LA-PCR} with betaine",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "6",
pages = "225-226",
month = "June",
year = "1997"}

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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