Multiplex PCR con Titanium Taq

TECH NOTE

Titanium Taq Outperforms Other Polymerases in High­Throughput Genotyping Multiplex PCR with Titanium Taq DNA Polymerase Data provided by Aaron Abbott, Helen Butler, Ph.D., Nicola Redhead, and Jiannis Ragoussis, Ph.D. Genomics Laboratory, Wellcome Trust Centre for Human Genetics

Overview The performances of three different brands of Taq polymerase were compared by evaluating the results of two MALDI­TOF single nucleotide polymorphism (SNP) analyses using the MassARRAY System from Sequenom. Titanium Taq DNA Polymerase performed better than the two alternatives in this type of high­ throughput analysis, indicating its suitability for demanding assays as well as everyday PCR. Titanium Taq DNA Polymerase tolerates a wide range of magnesium chloride (MgCl2) concentrations, amplifies targets of up to 2 kb from highly complex templates such as mammalian genomic DNA, amplifies rare or low­ copy targets, increases specificity using integrated hot start technology, and is optionally available in an eco­ friendly, lyophilized format (High Yield PCR EcoDry Premix).

More accurate results can be obtained more quickly with Titanium Taq DNA Polymerase.

Enzyme­Based, Whole­Genome SNP Genotyping Technologies enabling the detection and characterization (genotyping) of large numbers of SNPs from a large number of individuals allow identification of gene variants that underlie complex disorders. One high­throughput approach to whole­genome SNP genotyping is mass spectroscopy using platforms developed by Sequenom. In this highly accurate approach, allele­specific primers are used to perform primer extension reactions and the resulting products are compared by matrix­assisted laser­desorption/ionization­time­of­flight (MALDI­TOF) mass spectroscopy using the MassARRAY system (1). By amplifying and analyzing 96 or 384 samples in parallel, multiple alleles can be examined in many samples simultaneously.

Titanium Taq DNA Polymerase is a reliable, highly sensitive enzyme ideal for many applications including multiplex PCR. In the following assays, Titanium Taq DNA Polymerase produces the highest­quality SNPs (as measured by the overall pass rate and the number of SNPs that exceed an 80% pass rate threshold) and performs well over a range of MgCl2 concentrations. In the particular application studied, SNP genotyping using multiplex PCR, Titanium Taq DNA Polymerase accurately produces amplified SNPs that are of higher quality than those produced by the two other polymerases tested.

Minimal Optimization Required to Obtain Reliable Results

Maximizing the quality of the products of multiplexed PCR reactions depends on optimizing several factors: The accuracy of the polymerase The concentration of MgCl2 The concentration of the polymerase

These factors are particularly important to reliable detection of single base pair changes. TitaniumTaq removes these optimization barriers to reduce overall time expenditure. Here, we demonstrate that Titanium Taq provides accurate data (high SNP pass rate) and tolerates a range of MgCl2concentrations.

Three assays based on multiplexed PCR reactions were used to test the performance of TitaniumTaq DNA Polymerase: one nine­plex reaction (Figure 1), one six­plex reaction that was designed using Sequenom’s SpectroDESIGNER software, and one six­plex reaction that was designed manually.

First, the performance of Titanium Taq DNA Polymerase was evaluated at three MgCl2concentrations (2.5 mM, 3.5 mM, and 5.0 mM) and compared to the performances of Polymerases 1 and 2 at the recommended 2.5 mM concentration using the nine­plex reaction (Figure 1 and Table I). In one experiment, 576 genotypes were evaluated, with overall pass rates indicated in Table I.

Figure 1. Spectrum obtained using Titanium Taq DNA Polymerase in a nine­plex extension reaction (nine SNPs located on chromosome 16). The extension product peaks resulting from each SNP are color­coded and include the extension primer, two expected alleles, and a possible pausing peak. The peaks cover the 5,000–8,500 dalton range and are high quality.

Table I: Evaluation of DNA polymerases using nine­plex PCR DNA Polymerase

MgCl2 concentration

Overall pass rate

SNPs with <80% pass rate

Titanium Taq

2.5 mM

89.58

0

Titanium Taq

3.5 mM

90.74

0

Titanium Taq

5.0 mM

85.76

3

Polymerase 1

2.5 mM

86.00

1

Polymerase 2

2.5 mM

68.29

4

Table II: Evaluation of DNA polymerases using nine­plex PCR at 2.5 and 3.5 mM MgCl2 DNA Polymerase

MgCl2 concentration

Overall pass rate

SNPs with <80% pass rate

Titanium Taq

3.5 mM

95.80

0

High concentration Polymerase 2

2.5 mM

84.00

2

Polymerase 1

2.5 mM

85.76

2

Polymerase 2

3.5 mM

86.57

2

The far­right columns in Tables I and II indicate the number of SNPs with a pass rate lower than 80%, the quality threshold used in these experiments. In Table I, Titanium Taq DNA Polymerase had the highest overall pass rate at 3.5 mM MgCl2, closely followed by the same enzyme at 2.5 mM MgCl2.

Table II shows the results of a second experiment using the same nine­plex reaction shown in Figure 1. In this experiment, Titanium Taq DNA Polymerase in 3.5 mM MgCl2 (the best performer from Table I) was compared to Polymerase 1 in 2.5 mM MgCl2 and two concentrations of Polymerase 2; one concentration was comparable to the other polymerases in 3.5 mM MgCl2 and the other was a “high concentration” version in 2.5 mM MgCl2.

Based on the results of 500–800 individual reactions, the performance gap in favor of Titanium TaqDNA Polymerase was maintained regardless of MgCl2 and enzyme concentration. Again, TitaniumTaq DNA Polymerase demonstrated the highest overall pass rate of 95.8% at 3.5 mM MgCl2versus Polymerase 2’s best result pass rate of 86.57% at 3.5 mM MgCl2. The performance of Polymerase 2 did not improve even when 34% additional enzyme was used.

Consistent Performance in Demanding Assays Titanium Taq DNA Polymerase was compared to Polymerase 2 using two different six­plex reactions (Table III): one designed by hand (Reaction 1) and the other designed using Sequenom’s SpectroDESIGNER software (Reaction 2). Twelve different SNPs were analyzed, 96 DNA samples were tested, and the number of positive genotypes was determined (pass/96). The differences between Titanium Taq DNA Polymerase and Polymerase 2 were dramatic: Titanium Taq DNA Polymerase was consistently superior to Polymerase 2, which failed many of the individual SNP assays.

Table III: Evaluation of DNA polymerases using six­plex PCR SNP ID No.

Titanium Taq DNA Polymerase

Polymerase 2

Reaction 1

Pass/96

% pass

Pass/96

% pass

1

90

93.75

0

0.00

2

51

53.13

0

0.00

3

80

83.33

1

1.04

4

78

81.25

0

0.00

5

76

79.17

0

0.00

6

82

85.42

0

0.00

Reaction 2

26

93

96.88

4

4.17

30

94

97.92

2

2.08

31

87

90.63

74

77.08

33

91

94.79

65

67.71

40

91

94.79

0

0.00

50

89

92.71

0

0.00

Summary In these high­throughput, multiplex PCR assays for SNP genotyping, Titanium Taq DNA Polymerase emerged as the most reliable polymerase, showing consistently better performance than the other polymerases tested. In addition to improved yield and specificity, the researchers noted that Titanium Taq DNA Polymerase decreased the time needed for thermal cycling since it only requires a 1–2 minute activation time; other commercially available enzymes often require about 10–15 minutes. Overall, this led to a 14­minute reduction in cycling time, an important consideration for high­throughput applications.

Multiplex with confidence, sensitivity, power, and speed using Titanium Taq DNA Polymerase.

Methods Reactions were set up in 384­well plates at 5 µl total volume per reaction. The reaction mixture contained 2.5 ng of DNA, reaction buffer supplied by the manufacturer, dNTPs at 200 µM, Taqpolymerase at 0.1 unit (0.02 µl at 5 U/µl for all polymerases used), primers at 200 nM, and MgCl2 at the final concentration indicated in the tables.

In the second experiment with results in Table II, an additional 3.9 µl of Polymerase 2 was used in the "High concentration Polymerase 2" condition. PCR cycling conditions for Titanium Taq DNA Polymerase were: heating to 95°C followed by 45 cycles at 94°C for 20 sec, 56°C for 30 sec, and 72°C for 1 min followed by a final step at 72°C for 3 min. Initial heat activation was set at 15 min for Polymerase 1 and 10 min for Polymerase 2; otherwise, PCR conditions were identical.

References 1. Jurinke, C., et al. (2002) Methods Mol. Biol. 187:179–192.

http://www.clontech.com/US/Products/PCR_RT-PCR_Real-Time_qPCR/High_Yield_PCR_Polymerase/Titanium_Taq_for_Multiplexing