Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique Acknowledgement References
Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique
Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique
J. Powell*
The Richard Dimbleby Department of Cancer Research, I.C.R.F. Laboratory, Rayne Institute, 4th Floor Lambeth Wing, St Thomas's Hospital, Lambeth Palace Road, London SE1 7EH, UK
Received April 22, 1998;Revised and Accepted June 5, 1998
ABSTRACT
The Serial Analysis of Gene Expression (SAGE) method, described in 1995 by Velculescu et al., represents a powerful means to compare gene expression between two mRNA populations. An improvement to SAGE that removes contaminating linker molecules, which compromise the efficiency of the method, has been developed. This modification utilises biotinylated PCR primers, which generate biotinylated linkers at an early stage in the SAGE protocol, thus allowing removal of the unwanted linkers by binding to streptavidin-coated magnetic beads at a later stage. The application of this modification resulted in the rapid generation of high ditag yields and clones with large average insert sizes.
The Serial Analysis of Gene Expression (SAGE) method (1) generates short sequence tags which are positionally located within the cDNA molecule from which they are derived. This allows specific detection of that cDNA from a large number of different transcripts. The tags are generated as dimers, or ditags, and are ligated together to form concatemers which are then cloned. Sequencing the clones allows over 30 individual tags to be read from each lane of an automated sequencing gel. The abundance of a particular tag relates directly to the expression level of the gene from which it is derived. This serial analysis of many thousands of gene specific tags allows the simultaneous accumulation of information from genes expressed in the tissue of interest and gives rise to an expression profile of that tissue (1-5).
During the generation of the ditags linker molecules persist, despite a gel purification step, which have compatible sticky ends enabling them to ligate to the ditags (Fig. 1). This unwanted linker ligation terminates concatenation of that molecule and the reaction is effectively poisoned. The resulting molecule will not have compatible ends with which to clone into the prepared vector, pZero (Invitrogen). This leads to fewer clones being generated which, in turn, contain very few tags per clone. For SAGE to be efficient and to permit the analysis of large numbers of tags, the number of tags per clone must be as high as possible.
Figure1. Diagram showing how contaminating linkers are removed during the modification to the SAGE method. (a) Shown here are the products resulting from Step 8 of the detailed SAGE protocol (7). These are an unwanted 80 bp linker/linker artefact and the desired product, a 102 bp linker/ditag/linker molecule. (b) Bulk PCR reactions are performed using biotinylated primers A and B. Subsequent NlaIII digestion releases linkers and ditags as shown. The unwanted biotinylated linkers are removed by binding to streptavidin magnetic beads. (c) The remaining ditags are free from contaminating linkers and can ligate to form long clonable concatemers.
I describe a modification to the SAGE method which efficiently removes linkers from the ditag concatenation reaction and generates clones with large inserts and high tag numbers. In order to assess directly the improvement that this modification gives, a side by side comparison was carried out between the method I describe below and an alternative protocol which aims to remove the contaminating linkers using a gel-purification step, described by Velculescu et al. (6).
The modification is described as follows: biotinylated PCR primers are used to prepare bulk PCR reactions, (50 × 100 µl) of SAGE ditags, which have been produced exactly as described as in the detailed protocol (7). The 26 bp ditag sample is isolated without (at this stage) making any attempts to minimise linker contamination. As the linkers are now biotinylated, due to the PCR primers used in their generation, streptavidin coated magnetic beads (Dynabeads M-280 streptavidin, Dynal, Norway) can be used to extract all the biotinylated DNA as follows: the ditag sample is made up to 100 µl with LoTE (3 mM Tris-HCl pH 7.5, 0.2 mM EDTA pH 7.5). An aliquot of 100 µl 2× binding and washing buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2.0 mM NaCl) is then added, the sample divided in half and each half added to 100 µl streptavidin beads which have been pre-washed with 1× binding and washing buffer. After mixing, the samples are left at room temperature for 15 min with intermittent gentle agitation. A magnet is then used to immobilise the streptavidin beads/contaminating biotinylated linkers and the supernatant reserved. The beads are washed once with 1× binding and washing buffer and once with LoTE; the supernatant is reserved in each case. The supernatants are then ethanol precipitated and the pellets combined and resuspended in 7 µl LoTE. This purified ditag sample is then used in concatemer and clone formation, resuming the detailed SAGE protocol at step 11 (7).
For each method, the starting material consisted of 50 bulk PCR reactions of 100 µl, generated using biotinylated PCR primers (these were found to perform exactly the same as non-biotinylated primers; data not shown). The two methods were then carried out as described on each bulk PCR sample. Concatemers were generated and cloned. Several parameters were then determined for the SAGE libraries resulting from each method. The results are shown in Table 1.
For optimum performance of SAGE, maximum information is required from each clone sequenced in order to minimise the sequencing load per experiment. The amount of ditags generated is also critical to the cloning outcome, with several hundred nanograms of material being required for successful cloning of large concatenated inserts. Table 1 shows that, relative to the recent revisions of the originators of this technique (6), the method described here gave a greater yield of ditags. In addition, the average clone size was longer and the number of tags per clone was 43% greater, which increases the efficiency of the SAGE method.
These results therefore show that the modification described here represents a rapid and effective means of improving the efficiency of the SAGE method. Further SAGE libraries have been constructed in order to assess the reproducibility of the method. The results from these libraries also are presented in Table 1 and show a further improvement in clone size confirming reproducibility.
Ditags were generated using each protocol as described. After concatemer formation and cloning, 100 clones with inserts were analysed from the SAGE library resulting from each method. [The SAGE method was carried out using NlaIII as the anchoring enzyme and BsmFI as the tagging enzyme and the linkers described by Velculescu et al. (7). A clone insert consists of 226 bp of vector plus concatenated ditags, each of which is 26 bp. Each ditag represents two tags. Therefore, a clone of 616 bp equates to 30 SAGE tags, 226 bp of vector sequence plus 15 × 26 bp ditags.] Ditag yield describes the amount of ditags produced using each method, from a starting material of 50 identical bulk PCR reactions. The average clone insert size is an important parameter as large clone inserts are essential to the efficiency of SAGE. A single automated sequencing run can yield 600-1000 bp of readable sequence. Insert sizes approaching this range are therefore desirable. Experiment 1 describes results when SAGE was carried out using the modification described here. Experiment 2 shows the results obtained using the alternative method described by Velculescu et al. (6). Experiments 3-5 describe results from three further SAGE libraries constructed using the modification described here, showing that the technique gives a reproducible increase in clone size due to the effective removal of the contaminating linkers.
6. 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 Centre and Howard Hughes Medical Institute, 424 North Bond Street, Baltimore, MD 21231, USA; Fax: +1 410 955 0548.
7. Velculescu,V.E., Zhang, L., Vogelstein,B. and Kinzler,K.W. (1997) Serial Analysis of Gene Expression: Detailed Protocol (Version 1.0b), November 1995. Available as above.
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