- Email: [email protected]
Optimizing multiplex and LA-PCR with betaine
COMPUTER CORI ER
TIBS 2 2 - JU~VE 1 9 9 7
Methods and reagents O p t i m i z i n g m u ] t i p ! e x a n d LAoPCR w i t h b e t a i n e Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Intemet. 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 oa how ~o parta;',e in the newsgmup, see the accompanying box.
amplification of control sequence. Stalling can also occur during cycle sequencing reactions, which migh~ 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 KCi, or by the addition of co-solvents such as DMSO and g~yceml 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 BAFLing oesults One problem noted recently in the conditions without much success. They methods newsgroup is that, even though complain that some difficult DNAs just the sequence being screened for might will not amplify even when using some of be present in the template DNA, false the commercial kits designed for highnegatives can occur so that only the con- GC-content PCR. For example, one nettrol DNA band appears on a gel. Taq DNA ter wrote that after trying both the polymerase can stall within particularly Advantage-GC PCR kit from Clo,tech difficult regions of template DNA during and the Q-solution supplied with Qiagen's extension owing to the formation of sec- PCR kit, he still could not get a difficult ondary structures. This can cause low region to amplify. Also, addition of efficiency amplifications in the PCR, or tetramethylammonium chloride (TMAC) under conditions designed for multiplex as suggested by others Lz did not help. amplifications, the expected target DNA can be outcompeted by the more-efficient getaine Recently, someone questioned the use of a relatively new additive that can inservice from BIONET crease the amplification products from high-GC-containing sequences in multiThe latest messages posted to the bionet plex PCR. On the information sheet as well as all past archived messages are for LA-PCR provided by Wayne Barnes located at net.No.net and all you will need to do in order to read and/or post to any of (wayne@barnes 1.wustLedu) available the newsgroups is point your World W:de from http://mbb.wustl.edu/-barnes/faq. Web browser to the URL http://www.bio.net wpa, it is now recommended that 1.3 and then click on the 'Access the BIOSCI/ betaine (N,NJV-trimethylglycine; Sigma No. bionet Newsgroups' hypedink. B-2629) and 1.3% DMSO be added to LAA hypermail archiving system now gives you PCR mixtures to improve processivity3. the advantages of USENET without requiring Dr Barnes suggests that betalne be a local news server. The message headers added to LA-PCR, that the melting step of are threaded by default, but messages can each cycle should be reduced to 92-93°C, also be displayed chronologically or sorted by and the annealing temperature within the author or subject line. This capability gives you, in effect, a threaded newsreader through PCR cycles should be reduced by 1 or the Web. If you have any questions or en2°C to compensate for the change in ancounter any problems with the new server nealing conditions and some decreased please report them to [email protected] e n z y m e stability caused by the additive.
The technique of amplifying long stretches of DNA by the polymerase chain reaction (],A~PC~) 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 tempnate should be included for comparison to the ampiicon being tested.
Published by Elsevier Science Ltd. All rights reserved. 0968-0004/97
PII: 50968-0004(97)01069-4
He wrote that, not only are high-GCcontaining 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 betalne 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 quality4. 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 5on the use of 2 M betaine within sequencing reactions performed with doublestranded supercoiled plasmid DNA templates and T7 DNA polymerase (Sequenase~), 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 polymeruse, and that betaine somehow disrupts the contorted DNA helix without perturbing the polymerase-DNA interaction. It has been suggested that betalne 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~. 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 wrong dimensions In last month's column, a mistake was inadvertently introduced during the production process. The dimensions of the graphite blocks used on the home-made blotting aPparatus described should be i cm x 20 cm × 15 cm and not those stated. We apologize to Paul Hengen and to our readers mr this error. 225
COMPUTER C'()RNER
TIBS 22 = JUNE 1 9 9 7
.....
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 tenfold more expensive. Unfortunately, it appears that com#ar~ng 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).
Burningthe 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 hotter 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 cthidium bromide-stained agarose gel. After testing the s e p a r a t e 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 oxidationL Netters say that small volumes of oil (25-30 ml) should be irradiated only briefly and that adding 0.1% of the antioxidant 8-hydroxyquinoline before UV treatment can increase the life el your mineral oil stock, and it could actually help increase the yield of PCR product s. 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. (]_990) Nucleic Acids Res. 18. 4953 2 Chevet, E., Lemaitre. G. and Katinka. M. D. (1995) Nucleic Acids Res. 23, 3343-3344
Internet resources on G-protein-coupled receptors G-protein-coupled receptors I,a (GPCRs) metliato signals from a number of endogenous compounds, such as hormones, neurotransmitters, autocrine and paracrine factors, as well as having light-, odorant- and taste-sensing functions. GPCRs are also the target for a large number of drugs, and are therefore the object of intensive research world-wide. Although there is no published data at the atomic level available for any of these receptors, a variety of experimental and theoretical methods has validated a consensus wchitecture of an extracellular amino terrainus, cytoplasmic carboxy! terminus and seven transmembrane helices connected by loops for GPCRs ~. Several types of information and services on this important protein superfamily are summarized here (see also Table I and http://www-grap.fagmed. uit.no/farma/tibs_tablel.html, where a copy of the table, including links to the entries, can be found).
226
Seqaeace databases Although all aw,,ilable GPCR sequences can be found in general molecular biology sequence databases, SWiSS-PROT4 has some special features for this protein superfamily. SWlSS-PROT provides a complete and searchable list of GPCR sequences, organized into receptor classes, with links to the sequence database. Associated with SWISS-PROT is the SWISS-MODEL5 automated proteinmodelling server. For example, a threedimensional model of GPCR transmembrahe segments can be constructed by entering the mnino acid sequence for each transmembrane segment, and by choosing a three-dimensional template for the arrangement and packing of the transmembrane helices. The three-dimensional model of the transmembrane segments is returned to the user by Email. The Olfactory Receptor DataBase (OPDB) specializes in olfactory receptors, currently providing receptor sequences.
3 Baskaran. N. et al. (1996) Genome Res. 6, 633--635 4 Weissenstemer, T. and Lanci~bury, J. S. {.~996) BioTechniques 21, 1102-1108 5 Rees, W, A, et aL (1993) Biochemistry32, 137-144 6 Mytell~a, 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
PAUL N. HENGEN National Cancer Dnstitute, Frederick Cancer Research and Development Center, Frederick, MD 2 1 7 0 2 - 1 2 0 1 , USA. Email: pnh@ncifcr f.gov
-Any-statements -n;aOe g -the aatllor 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 necessariGy reflect the opinion of the National Cancer Institute or the National InstRutes of Health. An archive of Methods and reagents articles is available on the Internet and can be obtained by anonymous ftp from ftp.ncifcrf.gov in the directory pub/methods/TBBS, or on the World Wide Web from http://www-lmmb.ncifcrf.gov/-pnh/
Some el the access features are thnited to scientists working in the olfactory receptor field. Mutant databases Last year searchable mutant GPCR databases became available on the lnternet. The first mutant database '~was produced at the National Institutes of Health, Molecular Recognition Section (MRS, Table l) and had v e r y limited search facilities, although keyword searches are now possible. For each receptor domain (transmembrane helix, loop, etc.), one table of data can be retrieved. The second mutant database, Gprotein-coupled Receptor, family A, Point mutation database (GRAP), originating in our laboratory, has a more sophisticated search facility that allows searches for specific amino acid substitutions in specific protein domains within userspecified groups el receptors 7. This database also contains searchable information on quantitative ligand-binding data, and qualitative descriptions of the effect of the mutation(s) on agonist binding, antagonist binding and signal transduction. The GRAP mutant database can also be accessed from an amino acid sequence alignment. Because an enormous amo,~,t of work is required to computerize detailed data
Copyright © 1997.Elsevier Science Ltd. All rights reserved. 0968-0004/97/$17.00 PII:S0968-0004(97)01044-X
TIBS 2 2 - JU~VE 1 9 9 7
Methods and reagents O p t i m i z i n g m u ] t i p ! e x a n d LAoPCR w i t h b e t a i n e Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Intemet. 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 oa how ~o parta;',e in the newsgmup, see the accompanying box.
amplification of control sequence. Stalling can also occur during cycle sequencing reactions, which migh~ 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 KCi, or by the addition of co-solvents such as DMSO and g~yceml 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 BAFLing oesults One problem noted recently in the conditions without much success. They methods newsgroup is that, even though complain that some difficult DNAs just the sequence being screened for might will not amplify even when using some of be present in the template DNA, false the commercial kits designed for highnegatives can occur so that only the con- GC-content PCR. For example, one nettrol DNA band appears on a gel. Taq DNA ter wrote that after trying both the polymerase can stall within particularly Advantage-GC PCR kit from Clo,tech difficult regions of template DNA during and the Q-solution supplied with Qiagen's extension owing to the formation of sec- PCR kit, he still could not get a difficult ondary structures. This can cause low region to amplify. Also, addition of efficiency amplifications in the PCR, or tetramethylammonium chloride (TMAC) under conditions designed for multiplex as suggested by others Lz did not help. amplifications, the expected target DNA can be outcompeted by the more-efficient getaine Recently, someone questioned the use of a relatively new additive that can inservice from BIONET crease the amplification products from high-GC-containing sequences in multiThe latest messages posted to the bionet plex PCR. On the information sheet as well as all past archived messages are for LA-PCR provided by Wayne Barnes located at net.No.net and all you will need to do in order to read and/or post to any of (wayne@barnes 1.wustLedu) available the newsgroups is point your World W:de from http://mbb.wustl.edu/-barnes/faq. Web browser to the URL http://www.bio.net wpa, it is now recommended that 1.3 and then click on the 'Access the BIOSCI/ betaine (N,NJV-trimethylglycine; Sigma No. bionet Newsgroups' hypedink. B-2629) and 1.3% DMSO be added to LAA hypermail archiving system now gives you PCR mixtures to improve processivity3. the advantages of USENET without requiring Dr Barnes suggests that betalne be a local news server. The message headers added to LA-PCR, that the melting step of are threaded by default, but messages can each cycle should be reduced to 92-93°C, also be displayed chronologically or sorted by and the annealing temperature within the author or subject line. This capability gives you, in effect, a threaded newsreader through PCR cycles should be reduced by 1 or the Web. If you have any questions or en2°C to compensate for the change in ancounter any problems with the new server nealing conditions and some decreased please report them to [email protected] e n z y m e stability caused by the additive.
The technique of amplifying long stretches of DNA by the polymerase chain reaction (],A~PC~) 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 tempnate should be included for comparison to the ampiicon being tested.
Published by Elsevier Science Ltd. All rights reserved. 0968-0004/97
PII: 50968-0004(97)01069-4
He wrote that, not only are high-GCcontaining 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 betalne 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 quality4. 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 5on the use of 2 M betaine within sequencing reactions performed with doublestranded supercoiled plasmid DNA templates and T7 DNA polymerase (Sequenase~), 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 polymeruse, and that betaine somehow disrupts the contorted DNA helix without perturbing the polymerase-DNA interaction. It has been suggested that betalne 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~. 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 wrong dimensions In last month's column, a mistake was inadvertently introduced during the production process. The dimensions of the graphite blocks used on the home-made blotting aPparatus described should be i cm x 20 cm × 15 cm and not those stated. We apologize to Paul Hengen and to our readers mr this error. 225
COMPUTER C'()RNER
TIBS 22 = JUNE 1 9 9 7
.....
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 tenfold more expensive. Unfortunately, it appears that com#ar~ng 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).
Burningthe 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 hotter 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 cthidium bromide-stained agarose gel. After testing the s e p a r a t e 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 oxidationL Netters say that small volumes of oil (25-30 ml) should be irradiated only briefly and that adding 0.1% of the antioxidant 8-hydroxyquinoline before UV treatment can increase the life el your mineral oil stock, and it could actually help increase the yield of PCR product s. 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. (]_990) Nucleic Acids Res. 18. 4953 2 Chevet, E., Lemaitre. G. and Katinka. M. D. (1995) Nucleic Acids Res. 23, 3343-3344
Internet resources on G-protein-coupled receptors G-protein-coupled receptors I,a (GPCRs) metliato signals from a number of endogenous compounds, such as hormones, neurotransmitters, autocrine and paracrine factors, as well as having light-, odorant- and taste-sensing functions. GPCRs are also the target for a large number of drugs, and are therefore the object of intensive research world-wide. Although there is no published data at the atomic level available for any of these receptors, a variety of experimental and theoretical methods has validated a consensus wchitecture of an extracellular amino terrainus, cytoplasmic carboxy! terminus and seven transmembrane helices connected by loops for GPCRs ~. Several types of information and services on this important protein superfamily are summarized here (see also Table I and http://www-grap.fagmed. uit.no/farma/tibs_tablel.html, where a copy of the table, including links to the entries, can be found).
226
Seqaeace databases Although all aw,,ilable GPCR sequences can be found in general molecular biology sequence databases, SWiSS-PROT4 has some special features for this protein superfamily. SWlSS-PROT provides a complete and searchable list of GPCR sequences, organized into receptor classes, with links to the sequence database. Associated with SWISS-PROT is the SWISS-MODEL5 automated proteinmodelling server. For example, a threedimensional model of GPCR transmembrahe segments can be constructed by entering the mnino acid sequence for each transmembrane segment, and by choosing a three-dimensional template for the arrangement and packing of the transmembrane helices. The three-dimensional model of the transmembrane segments is returned to the user by Email. The Olfactory Receptor DataBase (OPDB) specializes in olfactory receptors, currently providing receptor sequences.
3 Baskaran. N. et al. (1996) Genome Res. 6, 633--635 4 Weissenstemer, T. and Lanci~bury, J. S. {.~996) BioTechniques 21, 1102-1108 5 Rees, W, A, et aL (1993) Biochemistry32, 137-144 6 Mytell~a, 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
PAUL N. HENGEN National Cancer Dnstitute, Frederick Cancer Research and Development Center, Frederick, MD 2 1 7 0 2 - 1 2 0 1 , USA. Email: pnh@ncifcr f.gov
-Any-statements -n;aOe g -the aatllor 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 necessariGy reflect the opinion of the National Cancer Institute or the National InstRutes of Health. An archive of Methods and reagents articles is available on the Internet and can be obtained by anonymous ftp from ftp.ncifcrf.gov in the directory pub/methods/TBBS, or on the World Wide Web from http://www-lmmb.ncifcrf.gov/-pnh/
Some el the access features are thnited to scientists working in the olfactory receptor field. Mutant databases Last year searchable mutant GPCR databases became available on the lnternet. The first mutant database '~was produced at the National Institutes of Health, Molecular Recognition Section (MRS, Table l) and had v e r y limited search facilities, although keyword searches are now possible. For each receptor domain (transmembrane helix, loop, etc.), one table of data can be retrieved. The second mutant database, Gprotein-coupled Receptor, family A, Point mutation database (GRAP), originating in our laboratory, has a more sophisticated search facility that allows searches for specific amino acid substitutions in specific protein domains within userspecified groups el receptors 7. This database also contains searchable information on quantitative ligand-binding data, and qualitative descriptions of the effect of the mutation(s) on agonist binding, antagonist binding and signal transduction. The GRAP mutant database can also be accessed from an amino acid sequence alignment. Because an enormous amo,~,t of work is required to computerize detailed data
Copyright © 1997.Elsevier Science Ltd. All rights reserved. 0968-0004/97/$17.00 PII:S0968-0004(97)01044-X