( ...)The use of TaqMaster PCR Enhancer

Guy Adami, Department of Oral Medicine, University of Illinois in Chicago, USA
Vincent Prezioso, Eppendorf Scientific, Inc., Westbury, NY, USA

Introduction

Transgenic mice carry a fragment of foreign DNA that has been incorporated into their genome. The most common method of introducing recombinant DNA into the mouse genome is by microinjection of the DNA into the pronuclei of one-cell stage mouse embryos.

A fraction of these embryos develop into newborn mice which have the micro-injected DNA stably integrated in their genome. A second strategy for generating transgenic mice involves genetic manipulation of ES cells.

After electroporation with a carefully designed construct, the stem cells are screened for the integration of new DNA through homologous recombination.

ES cells that are positive for a homologous recombination event are then transplanted into blastocyst stage embryos in order to generate chimeric mice. In most cases, gene targeting is used to introduce mutations into specific genes in the mouse genome.

The transgenic DNA is typically transcribed and translated in a tissue-specific and stage-specific pattern that is determined by regulatory sequences present in the transgenic DNA. Consequently, transgenic mice represent an experimental system in which it is possible to study the consequences and/or pattern of expression (or lack thereof) of the new genetic information. Incorporated transgenes are often detected by genomic DNA isolation from a tissue sample, followed by PCR* amplification of a region of the inserted gene.

The tissue source most commonly used for genotyping is mouse tail (1), however, ear, toe, hair, and blood samples are also used. Typically, genomic DNA isolation procedures from tail slices involve long incubations (one hour to overnight) at high temperature in the presence of Proteinase K. One of the problems that researchers often see is inconsistency in the amplification of transgenic DNA caused by impurities in the template DNA: These often inhibit PCR reactions causing false negative or inconclusive results. Here, we describe a reliable method of achieving consistent amplification of genomic DNA with impurities using a
novel PCR enhancer.

The Eppendorf MasterTaq Kit contains the TaqMaster PCR enhancer that has been specially developed to improve the amplification of difficult templates by improving the thermostability of the Taq DNA Polymerase. TaqMaster also adsorbs impurities in the template making the Taq less sensitive to exogenous PCR
inhibiting contamination. Impure and GC-rich templates show increased yields and reproducible results.

Results

Six newborn mice were screened for the presence of a TGF alpha transgene. Mouse tail DNA (from last 2 mm of tail) was isolated by Proteinase K digestion
followed by heat inactivation as described (2).

Briefly, 2 mm of tail was placed directly into 200 l DNA Extraction Buffer with Nonionic Detergents (PBND) (3) in a 1.5 ml microcentrifuge tube. Next 2.5 l Proteinase K solution at 20 mg/ml (4) was added to each sample.

The samples were incubated at 55 C with occasional vortexing until tissue was degraded (~2 hours). Finally, the samples were boiled for 10 minutes (to inactivate the Proteinase K), and centrifuged for 5 minutes. 2 l of this reaction was used per PCR reaction. The primers used in the reaction are specific to the human TGF alpha cDNA and were synthesized as described (5).

The size of the expected amplified product is 182 bp. The PCR was split into two sets of reactions, with and without the TaqMaster, according to the manufacturer's specifications (6). After an initial incubation at 94 C for 5 minutes, 0.2 l was added to each reaction. The PCR was run for 29 cycles under the following conditions:
1. Denature 94 C, 1 minute
2. Hybridization 56 C, 30 seconds
3. Extension 70 C, 40 seconds

Figure 1 shows the results of the experiment. All results were confirmed by phenotypic analysis of adult transgenic animals (7).



Figure 1. The TaqMaster enhancer makes the PCR signal both stronger and much more consistent. This is a 1.5 % agarose gel run with TAE. Lanes: 1 & 14 = 1 kb ladder (Life Technologies), Lanes 2-7 PCR without enhancer (TaqMaster), Lanes 8 -13 with the TaqMaster and the markers again.

The lanes with reactions that do not contain the PCR enhancer show variability and artifacts such as primer-dimers (lanes 2-7). Note especially lanes 3 and 4 where a weak positive signal could lead to ambiguity about the results. In contrast, lanes 8 -13 show extremely consistent results. Positive animals can be clearly identified as those corresponding to lanes 9, 10 and 13.

Note that lanes 9 and 10, which are from the same template as lanes 3 and 4, show robust amplification with no primer-dimers or other artifacts. The other lanes with the TaqMaster also show no primer-dimers and strong signals in the case of positive animals.

This demonstrates that the TaqMaster has the effect of making these reactions more robust and consistent.

Conclusion

Eppendorf's TaqMaster PCR enhancer is ideal for PCR reactions that contain templates with impurities.

References
(1) Hogan, B. Manipulating the Mouse Embryo 1994, p. 296

(2) Perkin Elmer Cetus: Vol.#2 PEC1989, pp. 1-3

(3) PBND buffer contains:
50 mM KCl
10 mM Tris-HCl (ph 8.3)
2.5 mM MgCl
0.1 mg/ml gelatin
0.45 % v/v Nonidet P40 (NP40)
0.45% v/v Tween 20

(4) Proteinase K: Made fresh for each use! 20 mg Proteinase K is dissolved in 1 ml ddH O @ 4 C. Filter sterilize.

(5) Ji H.J., Zhang Q.Q., Leung B.S. Survey of oncogene and growth factor/receptor gene expression in cancer cells by intron-differential RNA/PCR. 1990 Jul 31; 170 (2): pp. 569-575

(6) PCR reaction mix for ten tubes consists of: Rxn. with enhancer:
25 l 10x Buffer
50 l TaqMaster PCR Enhancer
20 l dNTP at 2.5 M
2.5 l Primer 1 at 1 g/l
2.5 l Primer 2 at 1 g/l
130 l H O
+2 l Tail DNA
(out of 200 l total per 2 mm piece of tail)
Rxn. without enhancer:
25 l 10x Buffer
20 l dNTP at 2.5 M
2.5 l Primer 1 at 1 g/l 20 mer
2.5 l Primer 2 at 1 g/l 20 mer
180 l H O
+2 l Tail DNA
(out of 200 l total per 2 mm piece of tail)

(7) Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. 1990 June 15; 61 (6): pp. 1137-1146


Applicational tip: PCR sample preparation