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Effective amplification of long targets from cloned inserts and human genomic DNA.
Open accessAbstract
We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.
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Cites
Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., & Erlich, H. A. (1988) · Science · doi:10.1126/science.2448875 · Cited by 17,719
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Cited by
Jeffreys, A. J., Kauppi, L., & Neumann, R. (2001) · Nature Genetics · doi:10.1038/ng1001-217 · Cited by 835
Stadhouders, R., Pas, S. D., Anber, J., Voermans, J., Mes, T. H. M., & Schutten, M. (2010) · The Journal of Molecular Diagnostics · doi:10.2353/jmoldx.2010.090035 · Cited by 399
Contributions of PCR-Based Methods to Plant Systematics and Evolutionary Biology
Wolfe, A. D., & Liston, A. (1998) · Molecular Systematics of Plants II · doi:10.1007/978-1-4615-5419-6_2 · Cited by 411
Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome
Vanzo, N. F., Li, Y. S., Py, B., Blum, E., Higgins, C. F., Raynal, L. C., Krisch, H. M., & Carpousis, A. J. (1998) · Genes & Development · doi:10.1101/gad.12.17.2770 · Cited by 299
Thalmann, O., Hebler, J., Poinar, H. N., Pääbo, S., & Vigilant, L. (2004) · Molecular Ecology · doi:10.1046/j.1365-294x.2003.02070.x · Cited by 285
Metadata
- Authors
- Cheng, S., Fockler, C., Barnes, W. M., & Higuchi, R.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 1994-06-07
- Volume
- 91
- Issue
- 12
- Pages
- 5695-5699
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2021243296
- Cited by
- 626
- References
- 1
- FWCI
- 20.22
- Type
- journal article
- Language
- en
- Open access
- green
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- Cheng, S., Fockler, C., Barnes, W. M., & Higuchi, R. (1994). Effective amplification of long targets from cloned inserts and human genomic DNA. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.91.12.5695
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