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Bodo Eickhoff and Jrgen van der Bosch
Experimental Immunopharmacology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany.
Tel.: +49 4537 188458 Fax: +49 4537 188404
email: jvdbosch@fz-borstel.de
Abstract
This application describes the use of a new commercial PCR enhancer for improving PCR efficiency: the TaqMaster. With this enhancer, an integral component of the MasterTaq Kit by Eppendorf, it was possible to amplify human hsp60 (heatshock protein) and hsp27 amplicons, an application in which five other polymerases, including hot start and proofreading enzymes, failed. The application of the PCR enhancer TaqMaster is highly recommended for solving problems with difficult targets, for detecting low copy RNAs or for increasing product yield.
Introduction
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a common method for the detection of gene expression. It can be used qualitatively to show principle expression of a specific target gene in a cell type or it may even serve to quantify the expression level of the gene of interest. In differential gene expression analysis, a search is undertaken for target genes that are regulated by certain conditions, eg, application of drugs. There are several methods for identifying these regulated genes; differential display RT-PCR,1 subtractive hybridization2 or complex hybridization onto cDNA arrays3 are some of the most common. With these techniques, a set of candidate sequences is obtained with or without a known identity. All these potentially regulated sequences need further confirmation that they really have been expressed differentially. One way of achieving this aim is by designing oligonucleotide primers to confirm the expression of the target sequence via RT-PCR in the cells under examination. If the RT-PCR is positive, the amplicon can be used as probe for detecting the corresponding mRNA on Northern blots or for semi-quantitative RT-PCR. As many sequences are low-copy genes and thus difficult to detect on Northern blots, RT-PCR is preferred in these cases. The results from subtractive hybridization in many cases consist of very short sequences (80250 bp), so the amount of possible PCR primers is limited. Nevertheless, to obtain any results from RT-PCR, a reliable amplification system is required.
Experimental setup
In the experiment described, the influence of an inhibitor of histone deacetylases on the gene expression of human lung adenocarcinoma cells was analyzed using subtractive hybridization4 of treated cells versus control cells. Of 36 oligonucleotide primer pairs designed to amplify candidate sequences, three failed to yield any PCR product with the standard Taq DNA Polymerase used for routine amplifications. PCR reaction conditions were modified using temperature gradient PCR (Eppendorf Mastercycler gradient)** for determining the optimal annealing temperature of the primers (50C70C), variation of MgCl2 concentration (1.5 mM to 5 mM) and touchdown PCR. None of these yielded any PCR product. Five different polymerases from other suppliers were then tested according to the manufacturers instructions, including two different proofreading polymerases and two hot start polymerases. Finally, the MasterTaq kit, containing a PCR enhancer, was used and resulted in positive RT-PCRs following the standard protocol given in the manual: Each 0.5 l of a reverse transcription reaction was used as template in the PCR reaction containing 0.6 M 5' primer, 0.6 M 3' primer, 0.2 mM dNTPs, 1 unit Taq DNA Polymerase and 10 l 5x TaqMaster PCR enhancer in a total volume of 50 l 1 x PCR buffer including MgCl2. This reaction mixture was amplified using the following temperature program: 96C 2 min and 30 cycles of 96C 5 sec, 58C 5 sec, 72C 15 sec.
Results and discussion
Positive RT-PCRs for hsp27 and hsp60 could only be performed using the MasterTaq Kit. As shown in Fig. 1, hsp60 (312 bp) and hsp27 (291 bp) were amplified with MasterTaq (A). However, amplification was insufficient and non-reproducible with Taq from another supplier (B). After direct sequencing of the hsp60 and hsp27 amplicons, they were labeled with [....32P] dATP for use as probes in a Northern blot experiment (Fig. 2). Using this strategy, it was possible to detect hsp60 and hsp27 mRNA in human lung tumor cells.
Figure 1: RT-PCR of hsp60 (312 bp) and hsp27 (291 bp), 10 l each loaded onto a 1.5% agarose gel (stained with ethidium bromide).Lane 1: 100 bp ladder (MBI Fermentas)
Lane 2: reverse transcriptase supplier 1/hsp60
Lane 3: reverse transcriptase supplier 2/hsp60
Lane 4: reverse transcriptase supplier 1/hsp27
Lane 5: reverse transcriptase supplier 2/hsp27
All reactions were of identical composition, except for enzyme and enhancer.
Series A: amplification using MasterTaq Kit (Eppendorf)
Series B: amplification using Taq Polymerase supplier M.
1: GeneRuler DNA ladder (MBI Fermentas)
2: display RT/hsp60
3: MMuLV RT/hsp60
4: display RT/hsp27
5: MMuLV RT/hsp27
A: Eppendorf MasterTaq 1u/50 l
B: MBI Fermentas TAQ 1 u/50 l
Figure 2: Northern blot of LT23 human lung adenocarcinoma cell RNA (8
g) hybridized with hsp60 and hsp27 probe, respectively (protocol
available from the authors). Image derived from Phosphorimager after 8
h exposure.
These results demonstrate the positive effect of TaqMaster on thermal
stability and processivity of Taq DNA Polymerase. The use of this PCR
enhancer dramatically improves RT-PCR sensitivity and allows the amplification
of target genes that otherwise prove difficult or impossible to be detected
via RT-PCR.
References:
- Liang P and Pardee AB. Differential display of eukaryotic messenger RNA
by means of polymerase chain reaction. Science. 1992:257(5072);967-71.
- Sagerstrm CG, Sun BI, Sive HL. Subtractive cloning: past, present,
and future. Annu Rev Biochem. 1997;66:751-83.
Soares MB. Identification and cloning of differentially expressed genes. - Curr Opin Biotechnol. 1997;8(5):542-6.
- Wang Z and Brown DD. A gene expression screen. Proc Natl Acad Sci. 1991;88:11505-9.
Fermentas is a registered trademark of AB Fermentas CORPORATION.
This product is sold under licensing arrangements with F. Hoffman-La Roche Ltd., Roche Molecular Systems, Inc. and Applied Biosystems.
*Practice of the patented polymerase chain reaction (PCR) process requires a license.
**The Eppendorf Thermal Cycler is an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents.
Mastercycler gradient (U.S. Pat. 6,210,958)
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