The efficiency of the original SAGE (Serial Analysis of Gene Expression) protocol was limited by a small average size of cloned concatemers. We describe a modification of the technique that overcomes this problem. Ligation of ditags yields concatemers of various sizes. Small concatemers may aggregate and migrate with large ones during gel electrophoresis. A heating step introduced before gel electrophoresis breaks such contaminating aggregates. This modification yields cloned concatemers with an average size of 67 tags as compared to 22 tags by the original protocol. It enhances the length of cloned concatemers substantially and reduces the costs of SAG

Kenzelmann, M.

Mühlemann, K.

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 1999 Oxford University Press 917–918Nucleic Acids Research, 1999, Vol. 27, No. 3Substantially enhanced cloning efficiency of SAGE(Serial Analysis of Gene Expression) by adding aheating step to the original protocolM. Kenzelmann* and K. MühlemannInstitute of Medical Microbiology, University of Bern, Friedbühlstrasse 51, 3010 Bern, SwitzerlandReceived October 16, 1998; Revised and Accepted December 1, 1998ABSTRACTThe efficiency of the original SAGE (Serial Analysis ofGene Expression) protocol was limited by a smallaverage size of cloned concatemers. We describe amodification of the technique that overcomes thisproblem. Ligation of ditags yields concatemers ofvarious sizes. Small concatemers may aggregate andmigrate with large ones during gel electrophoresis. Aheating step introduced before gel electrophoresisbreaks such contaminating aggregates. This modif-ication yields cloned concatemers with an average sizeof 67 tags as compared to 22 tags by the originalprotocol. It enhances the length of cloned concatemerssubstantially and reduces the costs of SAGE.The Serial Analysis of Gene Expression (SAGE) method (1)reduces cDNA molecules that have been reverse transcribed frompolyadenylated mRNA by a series of enzymatic manipulations totags of 10–13 bp. Each tag represents one mRNA. Tags are ligatedto form concatemers that are cloned and sequenced. Comparingthe sequence information from the tags with the GenBankdatabase provides qualitative information about transcribedgenes. The frequency of a specific tag within the SAGE tagpopulation correlates with its relative abundance in the cell andgives quantitative information about expressed genes. The SAGEmethod therefore allows for cataloging and comparison ofexpressed genes under various physiological conditions (1–7).The statistical analysis of the expression of thousands ofdifferent genes requires the sequencing of a large number of tags.Despite its elegant approach, the efficiency of the original SAGEprotocol was limited by a restriction of the length of clonedconcatemers to an average of ∼22 tags. Powell recently presenteda modified protocol which increased the number of tags per cloneby 43% from an average of 21 to 35 tags by the elimination ofcontaminating linker molecules (8). Instead of using unlabeledprimers as in the original SAGE protocol by Velculescu et al. (9),she used biotinylated primers to prepare bulk PCR reactions ofSAGE ditags. Ditags were then efficiently separated from thePCR primers by streptavidin coated magnetic beads instead of agel purification step. Here we describe another modification ofthe original SAGE protocol that increases the length of clonedconcatemers substantially.Referring to steps 10, 11 and 12 of the detailed SAGE protocol(9) bulk amounts of tags are obtained by PCR amplification of tagdimers, so called ditags (1 ditag = 26 bp). The linkers used tocreate these amplicons are removed after PCR amplification bya gel purification step. Purified ditags are then ligated to formconcatemers. The concatemers were separated according to sizeon an 8% polyacrylamide gel where they form a smear rangingfrom molecules of ∼300 bp to several kb in size. Cloning ofselectively cut out bands in the range of large size moleculesnevertheless yields inserts with an average size of 300 bp (∼22 tags).The reason for this size limit of clonable concatemers hasremained unclear. In our hands it was unaffected by the use of theDNA condensing agent hexammine cobalt(III) chloride (data notshown).We modified the protocol as follows. The concatemerizationreaction of ditags (∼300 ng of DNA per reaction) was performedas described in the original protocol (step 11) with the exceptionthat we incubated the ligation reaction overnight. The ligationsample was then heated for 15 min at 65C and quickly chilledon ice for 10 min. Concatemers were separated on an 8%polyacrylamide gel as described above. This heating step shiftedthe banding pattern in the gel towards concatemers of smallersizes (Fig. 1). We compared the effect of the heating step on thecloning efficiency by dividing the sample into three size fractions:fraction 1 ranging from 700 to 1000 bp; fraction 2 comprisingmolecules of 1000 to 1600 bp; and fraction 3 ranging from 1600to 2500 bp. Following the protocol of Velculescu et al. (9), weobtained clones with average insert sizes of ∼300 bp (∼22 tags perclone; Table 1). These results are in good agreement with thoseobtained by other groups (7,8). As demonstrated in Table 1,heating of concatemers before gel separation led to a substantialincrease in the length of cloned concatemers from an average of287 bp (22 tags) to 873 bp (67 tags).*To whom correspondence should be addressed. Tel: +41 31 632 3251; Fax: +41 31 632 3550; Email: kenzel@imm.unibe.ch Nucleic Acids Research, 1999, Vol. 27, No. 3918Table 1. Comparison of length of cloned concatemers obtained by the original and the modified SAGE protocolAverage concatemer size [number of tags per clone]Non-heated samplea Heated samplebFraction 1 (700–1000 bp) 137 (± 47) [11 (± 3)] 669 (± 119) [51 (± 10)]Fraction 2 (1000–1600 bp) 208 (± 100) [16 (± 8)] 993 (± 342) [76 (± 27)]Fraction 3 (1600–2500 bp) 542 (± 105) [42 (± 8)] 1455 (± 225) [112 (± 17)]Average of fractions 1–3 287 (± 201) [22 (± 16)] 873 (± 346) [67 (± 27)]Average results from two independent cloning experiments. Fractions 1–3 represent concatemer size ranges that were selectivelycut out of the 8% polyacrylamide gel (step 11 of the detailed protocol) as outlined in the text. The average concatemer size reflectsthe length of the cloned concatemers without vector sequence (226 bp; vector pZErO, Invitrogen). Numbers in round bracketsare standard deviations.aResults were obtained by following the detailed SAGE protocol of Velculescu et al. (9). The average percentage of clones thatcontained no insert or an inappropriately small insert size was ∼30% in all three fractions.bResults were obtained using the modified protocol described in the text. The average percentage of clones that contained no insertor an inappropriately small insert size was 20–30% for fraction 1, 40–50% for fraction 2 and ∼70% for fraction 3. The increasingproportion of false positives that parallels the increasing length of cloned DNA fragments is a common observation in cloningexperiments and is most probably due to an increasing rate of vector self ligation.Figure 1. Separation of concatemers by 8% polyacrylamide gel electrophoresisbefore cloning [step 11 of the original protocol by Velculescu et al. (9)].Approximately 300 ng of gel purified ditags were used for the concatemerizationreaction. Lane 1, non-heated concatemer sample according to the originalprotocol of Velculescu et al. (9); lane 2, heated concatemer sample; lane 3,100 bp ladder molecular marker (GenSura). The DNA was stained withSYBR Green I (FMC BioProducts). Heating the concatemer sample leads toa shift in the banding pattern towards molecules of smaller size.Our results suggest that the ligation of ditags to concatemersmay be incomplete, yielding a large fraction of fragments of smallsize. These fragments may aggregate by hydrogen bonds betweenthe 4 bp overhangs created by cleavage with the anchoringrestriction enzyme NlaIII. The hydrogen bonds may be furtherstabilized by the high amount of Mg2+ ions in the ligation buffer(final concentration: 10 mM MgCl2; 5× ligase buffer, Gibco BRLLife Technologies) and by inactivated enzyme molecules bindingto the overhang sites of the concatemer fragments. These aggregatesmigrate through the gel as apparently large concatemer molecules.During cloning they disaggregate to their original smaller size.Heating the ligation sample before gel electrophoresis breakshydrogen bonds and prevents contamination of the large sizefraction by aggregates of smaller molecules. The same effect mayalso be obtained in other experiments that involve ligationreactions and cloning.For an optimal performance of SAGE (high throughput of tagswith minimal sequencing costs) it is essential to obtain maximuminformation per clone. Powell (8) recently described a modificationthat overcomes the problem of contaminating linker moleculesand increases the amount of tags per clone from an average of 21 to35. Our modification of the SAGE protocol addresses theincomplete ligation during the concatemerization process andleads to an even greater increase of the number of tags per cloneto 67. Both modifications may act synergistically if combined.The integration of Powell’s and our modification into the originalprotocol of Velculescu et al. (9) optimizes the efficiency of SAGEby reducing the number of sequencing reactions.ACKNOWLEDGEMENTSWe thank B. Fartmann, J. Graf, T. Seebeck, R. Friis and M. Täuberfor technical advice, valuable discussion, encouragement andsupport.REFERENCES1 Velculescu,V.E., Zhang,L., Vogelstein,B. and Kinzler,K.W. (1995) Science,270, 484–487.2 Velculescu,V.E., Zhang,L., Zhou,W., Vogelstein,J., Basrai,M.A., Bassett,D.E.,Hieter,P., Vogelstein,B. and Kinzler,K.W. (1997) Cell, 88, 243–251.3 Zhang,L., Zhou,W., Velculescu,V.E., Kern,S.E., Hruban,R.H., Hamilton,S.R.,Vogelstein,B. and Kinzler,K.W. (1997) Science, 276, 1268–1272.4 Madden,S.L., Galella,E.A., Zhu,J., Bertelsen,A.H. and Beaudry,G.A.(1997) Oncogene, 15, 1079–1085.5 Polyak,K., Xia,Y., Zweier,J.L., Kinzler,K.W. and Vogelstein,B. (1997)Nature, 389, 300–305.6 Hermeking,H., Lengauer,C., Polyak,K., He,T., Zhang,L., Thiagalingam,S.,Kinzler,K.W. and Vogelstein,B. (1997) Mol. Cell, 1, 3–11.7 Bertelsen,A.H. and Velculescu,V.E. (1998) Drug Discov. Today, 3, 152–159.8 Powell,J. (1998) Nucleic Acids Res., 26, 3445–3446.9 Velculescu,V.E., Zhang,L., Vogelstein,B. and Kinzler,K.W. (1997)Serial Analysis of Gene Expression: Detailed Protocol (Version 1.0c),September 1997. Available from Johns Hopkins Oncology Center andHoward Hughes Medical Institute, 424 North Bond Street, Baltimore, MD21231, USA; Fax: +1 410 955 0548.

Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) by adding a heating step to the original protocol

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