( Simultaneous purification of 96 P...)High-Throughput System Generates Ultra-Pure PCR Products Suitable for All,,,Applications

Simultaneous purification of 96 PCR products

Scott Basehore Jeff Braman
Stratagene

Andrew Firmin
Phenogenex

The StrataPrep 96 PCR purification kit is a new member of the StrataPrep product line. This kit offers a high-throughput format for purifying ultra-pure PCR product DNA from large numbers of samples. The StrataPrep 96 PCR purification kit uses the same silica matrix-binding technology as the other StrataPrep Kit products, but in a 96-well plate format. A vacuum manifold and centrifuge are used to recover 5 g per well of DNA ranging in size from approximately 100 bp to 23,000 bp. Purified PCR products are free of PCR artifacts as well as contaminants from adjacent wells and oil used in some PCR protocols. The purified PCR products are at an ideal concentration for automated fluorescent dideoxynucleotide sequencing. Better sequence data was obtained with a template purified with the StrataPrep 96 PCR kit, compared to the same PCR template with another commercially available kit.

In 1997, Stratagene introduced the StrataPrep PCR purification kit¹, an effective miniprep-scale tool for purifying PCR products in applications such as cloning and automated fluorescent dideoxynucleotide sequencing². The basis of this successful product is its unique glass fiber matrix, which captures DNA in the presence of a chaotropic salt. With the kits wash solution, unincorporated amplification primers and nucleotides, buffer components, enzymes, and nonspecific amplification products smaller than 100 bp can be removed from the matrix-bound PCR products. To recover the desired PCR products, low-ionic-strength buffer is added to the fiber matrix. This step is followed by centrifugation to collect the DNA at a concentration that is suitable for many applications.

Fig.1

For many disciplines, including molecular biology, biochemistry, neuroscience, and clinical diagnostics, the need to screen and process many PCR products simultaneously has significantly increased. Stratagene has responded to this need by transforming the StrataPrep Kit technology into a high-throughput format, making it possible to process as many as 96 samples simultaneously. In the StrataPrep 96 PCR purification procedure (Figure 1), an equal volume of DNA binding solution is added directly to PCR reactions (with or without an oil overlay). The entire mixture is then transferred to a StrataPrep 96 Kit binding plate. After washing to remove contaminants, PCR products are eluted in a small volume of low-ionic-strength buffer and are ready for many applications such as restriction digestion and automated fluorescent dideoxynucleotide sequencing.

The StrataPrep 96 PCR purification kit includes 96-well binding plates, 96-well collection and waste plates, plate sealers, storage mats, and all necessary buffers. As an added convenience, the kits components are compatible with most 96-well plate vacuum manifolds and 96-well-adapted centrifuges and can be adapted to a robotic system, such as the Biomek 2000 workstation (Beckman Coulter)

Ninety-Six Independent Samples

The 96-well plates have innovative drip directors that channel liquid flow out of the wells into corresponding wells of collection plates. To demonstrate that these drip directors eliminate well-to-well contamination between samples, 2.3-kb PCR products were purified in alternate wells of a StrataPrep 96 PCR purification plate. The plate was processed as if all wells contained PCR products, and samples from each well were visualized on an agarose gel. As seen in Figure 2, the wells that were processed without samples show no contamination products from adjacent wells. Furthermore, the ethidium bromide staining contaminant present in the unpurified sample, migrating in advance of the 250-bp marker (Lane 2), is eliminated from wells of the StrataPrep 96 plate (Lanes 3, 5, 7, 9, 11, and 13).

Fig.2

Consistent Results in Variable Conditions

The performance of the StrataPrep 96 PCR kit was evaluated by first determining the DNA binding capacity of the kits binding plate. Increasing quantities of linear double-stranded plasmid DNA were loaded into plate wells. The wells were washed, DNA was eluted, and the recovered DNA was quantitated by UV spectrophotometry (data not shown). The maximum amount of DNA recovered was 5 g per well. The size range of DNA recovered was also determined by loading a mixture of linear double-stranded DNA ranging in size from 115 bp to 23,000 bp into each well of the binding plate. The plate was washed, DNA was eluted, and DNA recovery from 15 randomly chosen wells was assessed by gel electrophoresis. Figure 3 shows uniform recovery of all DNA sizes from the wells of the plate.

Fig.3

One time-consuming step in most purification schemes is removing the oil overlay from amplification reactions. Although this step is a nuisance, it is often essential for recovering PCR products of sufficient purity for subsequent applications. Without modifying the standard purification procedure, this variable was examined by purifying two 50-l amplification reactions, one with and one without the oil overlay. An equal volume of DNA binding solution was added directly to both PCR mixtures, and were loaded into adjacent wells of a StrataPrep 96 PCR binding plate. The wells were washed, and the PCR products were eluted. Figure 4 shows quantitative and equivalent recovery of a 229-bp PCR product. The unpurified PCR product is shown in Lane 2 to compare recovery of the purified samples. The purified products in Lanes 3 and 4 were from identical amplifications with and without an oil overlay, respectively. This data demonstrates that the samples of varying condition in Lanes 2, 3, and 4 are equally well purified. The resulting purified DNA was successfully digested to completion with a variety of restriction enzymes (Figure 4, Lanes 5 through 14).

Fig.4

Highly Pure DNA Ideal for Automated Sequencing

PCR products were amplified from a plasmid DNA template and purified with either the StrataPrep 96 PCR purification kit or Qiagens QIAquick 96 PCR purification kit. The purified samples were sequenced with the automated fluorescent dideoxynucleotide method, and the data were compared. Figure 5 shows that the StrataPrep 96 PCR kit resulted in highly purified DNA with sequence data showing low-background, high-signal intensity data with no ambiguities in over 500 bases. In contrast, the QIAquick 96 PCR kit demonstrated sequence data that had high background, variable signal intensity, and numerous ambiguities.

Fig.5

Conclusions

The StrataPrep 96 PCR purification kit is a fast and efficient system for generating ultrapure DNA in a high-throughput format, processing of as many as 96 samples. This kit uses the same successful principles of a silica fiber-matrix binding membrane as other DNA purification products in the StrataPrep family. The StrataPrep 96 PCR purification protocol purifies DNA in a small, ready-to-use elution volume ideal for automated sequencing and other applications requiring highly purified PCR product. The StrataPrep 96 PCR purification kit represents a timesaving convenience and is a complete kit that includes all necessary 96-well plates, sealers, mats, and buffers.

REFERENCES

1. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.

2. Braman, J. and Basehore, S. (1999) Strategies 10: 84.