( A streamlined a...)Purified Single-Stranded M13 DNA for Automated Sequencing

A streamlined and reliable method to obtain high-quality single-stranded DNA

Jeff Braman Scott Basehore
Stratagene

The StrataPrep M13 DNA purification kit allows easy, quick, and reliable purification of bacteriophage single-stranded DNA. The yield of single-stranded DNA recovered from phage lysate exceeds the yield obtained with the leading commercial silica-gel-based (SGB) M13 DNA purification method. The resulting high-purity DNA is ideal for automated sequencing applications.

M13 DNA is a preferred template for many sequencing projects.^1-5 For many years, the standard laboratory protocol used to purify M13 DNA relied upon polyethylene glycol (PEG)-NaCl precipitation of phage particles, phenol-chloroform extraction to remove phage coat proteins, and ethanol precipitation to concentrate the DNA.⁶ This method is time-consuming, employs hazardous chemicals, and is difficult to apply to more than a few samples.

However, with the StrataPrep M13 DNA purification kit, isolating high-purity M13 DNA is possible without these cumbersome steps because the procedure is streamlined: Collect M13 phage particles on the unique glass fiber matrix contained in a StrataPrep spin cup;⁷ add a chaotropic salt solution to the spin cup so the phage coat protein denatures and binds the liberated single-stranded DNA to the glass matrix; centrifuge-wash the phage DNA to remove contaminating protein and salt; and elute the DNA from the spin cup with a low ionic strength buffer. The concentration of the highly purified DNA that results is ideal for automated sequencing applications.

High Yield Recovery of Single-Stranded DNA

The yield of single-stranded DNA from StrataPrep M13 method of Phagescript lysate (a modified M13 bacteriophage⁸), was at least twice that obtained from the SGB method (Figure 1). For 3 ml of phage lysate, the StrataPrep method yielded 7.5 g of DNA at a concentration of 25 ng/l, as judged by gel electrophoresis.

Figure 1

Quality Automated Sequencing

Equal volumes of StrataPrep- and SGB-purified Phagescript DNA were subjected to fluorescent dye primer automated sequencing by an independent sequencing service (Sequetech Corp.; Figure 2). Signal intensity was more than twice as high for the StrataPrep-purified template (Figure 2, Panel A) compared to the SGB-purified template (Figure 2, Panel B). Sequence read length was nearly 700 bases for both templates. There were three and four sequence ambiguities scored for the StrataPrep- and SGB-purifed templates, respectively, within the first 20 nucleotides. Sequence ambiguities also occurred at nucleotides 553 and 638 for the StrataPrep-purified template and at nucleotide 44 for the SGB-purified template.

Figure 2

Conclusion

The StrataPrep M13 DNA purification kit contains a fast, easy, and reliable method for M13 purification. Recover single-stranded DNA from phage lysate more efficiently using the StrataPrep method than with the leading commercial silica-gel-based method, and realize high-quality automated fluorescent sequencing results as well. Additionally, the kit can be used to purify single-stranded DNA from rescued phagemid clones.

REFERENCES

1. Richards, S., et al. (1994) In Automated DNA Sequencing and Analysis (Eds. Adams, M.D., et al.) pp. 191-198. Academic Press, San Diego.
2. Wilson, R.K. and Mardis, E.R. (1997) In Genome Analysis: A Laboratory Manual (Eds. Birren, B., et al.) Vol.1, p. 434. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
3. Blattner, F.G., et al. (1997) Science 277: 14531462.
4. Ali Ansari-Lari, M., et al. (1998) Genome Res. 8: 29-40.
5. Anderson, B., et al. (1998) Genome Res. 8: 809-816.
6. Maniatis, T., Fritsch, E., and Sambrook, J. (1982) In Molecular Cloning: A Laboratory Manual, Second Edition. pp. 4.29-4.30. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
7. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.
8. Stratagene Catalog (1999) p. 65.