Significantly Improve PCR Product Yield From Plasmid And Genomic DNA Templates

YieldAce™ DNA polymerase: the DNA polymerase with more output than Taq

Significantly Improve PCR Product Yield from Plasmid and Genomic DNA Templates

Michael Borns • Holly Hogrefe

Stratagene

YieldAce ^™ DNA polymerase *^,‡ is a proprietary enzyme formulation specifically developed for maximizing PCR product yields. We demonstrate the exceptional performance of this enzyme, in comparison to Taq DNA polymerase, by amplifying a large set of I.M.A.G.E. consortium cDNA clones. With YieldAce DNA polymerase, 99.5% of the clones tested (1 failure per 192 clones attempted) were successfully amplified, and 97.4% of the reactions produced single bands. Moreover, high product yields were consistently generated for all insert sizes (0.1-2.5 kb). In comparison, PCR product yields were 2.4- to 3-fold lower in reactions performed with Taq DNA polymerase. YieldAce DNA polymerase also produces higher product yields than Taq DNA polymerase in PCR comparisons employing genomic DNA templates.

Stratagene’s YieldAce DNA polymerase is a new enzyme formulation specifically developed for maximizing PCR product yields. This proprietary enzyme has been optimized to provide superior performance in a variety of applications routinely carried out with Taq DNA polymerase. Compared to Taq DNA polymerase, YieldAce DNA polymerase synthesizes higher yields of product from a variety of DNA templates, including genomic DNA, and cDNA, and it readily amplifies templates directly from bacterial cultures.  Additionally, reactions performed with YieldAce DNA polymerase show less variation in product yield with respect to amplicon size and DNA template concentration, than identical reactions carried out with Taq DNA polymerase.

PCR Protocols for Microarrays

One application that demands a robust and reliable PCR enzyme is the production-scale amplification of clone collections for microarrays.^1,2,3 To create a microarray, DNA fragments representing the collection of genes to be assayed are amplified by PCR and then robotically spotted at high density on glass slides.

Inserts are amplified from purified plasmid DNA or, more preferably, directly from bacterial cultures. Typically, PCR protocols are optimized for each library and primer set, and the same protocol is used for the entire library. For maximum efficiency, the PCR protocols developed for microarray production should successfully amplify most or all of the sequences in the clone collection.Amplification procedures should generate high yields of specific products with no background bands. Achieving high yield and specificity facilitates purification procedures, the preparation of multiple arrays, and arraying at high DNA concentrations (0.3-0.5 mg/ml). For clone collections containing larger inserts, PCR protocols should also synthesize high yields of longer products (>1 kb).

Amplification of cDNA Clone Collections with YieldAce^™ DNA Polymerase

To illustrate the superior yield capability of YieldAce DNA polymerase, we amplified plasmid inserts from hundreds of I.M.A.G.E. consortium cDNA clones (Research Genetics). Inserts ranging from approximately 0.1 kb to 2.5 kb in length were amplified from bacterial cultures provided in two arbitrarily selected 96-well plates. PCR reactions (50 µl) employed 2.5 U of YieldAce DNA polymerase, 0.5 µl of each bacterial culture, and 300 µM of each vector-specific primer (Research Genetics).

The optimal temperature cycling conditions for YieldAce DNA polymerase are as follows: (segment 1) 1 cycle at 92°C for 2 minutes, (segment 2) 10 cycles of 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 2 minutes 30 seconds, (segment 3) 20 cycles of 95°C for 20 seconds, 55°C for 20 seconds, and 72°C for 2 minutes 30 seconds with an incremental increase in extension time of 10 seconds per cycle, (segment 4) 1 cycle of 72°C for 7 minutes. An initial denaturation temperature step of 92°C for 2 minutes provided sufficient DNA template denaturation with minimal thermal damage. In addition, the cycling profile was divided into two segments (segments 2 and 3), each with a different annealing temperature and extension time. In segment 2, high priming specificity is maintained by using a relatively high annealing temperature and a short hold time. In segment 3, efficient amplification is favored by reducing the annealing temperature and increasing the extension time.

Table 1
Success Rate for Amplifying cDNA Clones with YieldAce™ DNA Polymerase

Table 1 summarizes results obtained with YieldAce DNA polymerase using optimized cycling conditions. Of the 192 clones tested, 99.5% were successfully amplified with YieldAce DNA polymerase (1 failure per two 96-wells plates). Moreover, 97.4% of the successful reactions produced a single band (data not shown).

Improved DNA Yield from Clone Collections Greatly Reduces the Cost per Microarray

Fig.1

The superior yield produced by YieldAce DNA polymerase was demonstrated in side-by-side comparisons with Taq DNA polymerase. The cDNA clones described above were amplified with Taq DNA polymerase under identical conditions (2.5 U of enzyme; 0.5 µl of bacterial culture) using either the manufacturer’s recommended cycling conditions (Methods) or the optimal cycling conditions for YieldAce DNA polymerase. Figure 1 shows representative results for 24 cDNA clones from two rows of the same 96-well plate using conditions optimized for YieldAce for both enzymes. YieldAce DNA polymerase produced higher product yields than Taq DNA polymerase for all clones tested.  When Taq DNA polymerase amplifications were carried out under conditions optimized for Taq, YieldAce again outperformed Taq in product yield (see below).

Product yields obtained for the 24 representative cDNA clones (shown in Figure 1) were quantitated using the PicoGreen^® dsDNA Quantitation Kit (Molecular Probes). Under the same cycling conditions (YieldAce conditions), an average of 2.4-times more PCR product was produced with YieldAce DNA polymerase compared to Taq DNA polymerase (data not shown). When compared using the recommended conditions for each enzyme, PCR product yields were an average of three times higher for reactions performed with YieldAce DNA polymerase compared to Taq DNA polymerase (Figure 2).

Fig.2

An average 3-fold improvement in yield can result in a dramatic cost savings when printing microarrays.  Stratagene has estimated that the use of YieldAce instead of Taq DNA polymerase can save over $30 (U.S. dollars) per microarray of 10,000 spots.  The cost savings are the result of synthesizing more DNA with 2.5U YieldAce DNA polymerase, which allows more microarray slides to be printed.  There are many fixed costs in carrying out PCR reactions (clone growth, DNA purification, primer cost, plasticware, labor, and overhead) that are divided amongst the number of slides that can be printed.  Because YieldAce DNA polymerase generates more PCR product, more slides can be printed and therefore the fixed costs per slide are much less. Please see Stratagene’s web site at www.stratagene.com for a sample calculation.

Figure 3 shows enzyme comparisons for the 13 most difficult-to-amplify clones identified in the two 96-well plate set (192 clones). The reactions shown were produced with YieldAce and Taq DNA polymerases, using the recommended cycling conditions for each enzyme. The enzymes exhibited comparable specificity (single-band purity for 12/13 reactions), but YieldAce polymerase produced significantly higher product yields than Taq DNA polymerase (Figure 3).  Higher yields were also seen with YieldAce DNA polymerase when amplifying purified plasmid DNA (data not shown).

Fig.3

Improved Amplification of Genomic DNA Compared to Taq DNA Polymerase

We also compared YieldAce DNA polymerase to Taq polymerase in amplifications of genomic DNA targets (Figure 4). PCRs were performed under identical conditions, using each enzyme’s recommended buffer (Methods). YieldAce DNA polymerase consistently produced high yields of all genomic DNA targets, ranging in length from 0.1 kb to 2.6 kb. In comparison, Taq DNA polymerase synthesized each amplicon in lower yield (Figure 4).

Fig.4

Conclusion

YieldAce DNA polymerase excels in procedures routinely carried out with Taq DNA polymerase. Compared to Taq, YieldAce DNA polymerase produces higher product yields from both plasmid and genomic DNA templates. Its superior yield capability and reliability make it the enzyme of choice for production-scale amplification of clone collections for microarrays. Using YieldAce DNA polymerase reduces or eliminates time-consuming laborious steps such as extensive optimization studies, repeating failed PCRs, and pooling multiple PCRs to achieve high product yields. Finally, the enzyme’s high yield capability ensures uniform product yield for all insert sizes.

Methods

PCR reactions (50 µl) were assembled at room temperature and consisted of  200 µM each dNTP, 300 µM each primer (Research Genetics; GF200), and either 0.5 ml of bacterial culture (Research Genetics; I.M.A.G.E. cDNA clones) or 100 ng of genomic DNA.  Amplifications were carried out using 2.5 U of YieldAce DNA polymerase or AmpliTaq DNA polymerase in the recommended PCR buffer.

Reactions were cycled in 200-ml thin-walled PCR tubes using a Applied Biosystems 9600 single-block thermocycler. Enzyme comparisons employed optimized temperature cycling conditions for YieldAce and AmpliTaq DNA polymerases. Conditions optimized for YieldAce DNA polymerase are as follows: (segment 1) 1 cycle at 92°C for 2 minutes; (segment 2) 10 cycles of 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 2 minutes 30 seconds; (segments 3) 20 cycles of 95°C for 20 seconds, 55°C for 20 seconds, and 72°C for 2 minutes 30 seconds, with an incremental increase in extension time of 10 seconds per cycle. Conditions recommended by Research Genetics for Taq DNA polymerase are as follows: (segment 1) 1 cycle at 96°C for 30 seconds; (segment 2) 30 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 2 minutes 30 seconds. For each cycling profile, one final extension cycle of 72°C for 7 minutes was performed (after 30 cycles).

PCR products were electrophoresed on 1% agarose/1X TBE gels and visualized by ethidium bromide staining. The gels were imaged using the Eagle Eye^® II still video system.

PCR reactions were quantitated using the PicoGreen dsDNA Quantitation Kit (Molecular Probes). PCR reaction dilutions were prepared (following kit’s protocol) to fall within the linear detection range of the PicoGreen assay using the high-range standard curve. Absorbencies were measured using a Wallac 1420 fluorescent microplate reader.

REFERENCES

1. Ramsey, G. (1998) Nat. Biotechnol. 16: 40-44.
2. Ferea, T.L. and Brown, P.O. (1999) Curr. Opin. Genet. Dev. 9: 715-722.
3. Duggan, D.J. et al. (1999) Nat. Genet. 21: 10-14.

* U.S. Patent Nos. 5,545,552, 5,556,772, 5,866,395 and 5,948,663 and patents pending