Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jul 1;25(13):2657–2660. doi: 10.1093/nar/25.13.2657

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

I Afonina 1, M Zivarts 1, I Kutyavin 1, E Lukhtanov 1, H Gamper 1, R B Meyer 1
PMCID: PMC146788  PMID: 9185578

Abstract

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.

Full Text

The Full Text of this article is available as a PDF (102.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Afonina I., Kutyavin I., Lukhtanov E., Meyer R. B., Gamper H. Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3199–3204. doi: 10.1073/pnas.93.8.3199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asseline U., Delarue M., Lancelot G., Toulmé F., Thuong N. T., Montenay-Garestier T., Hélène C. Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3297–3301. doi: 10.1073/pnas.81.11.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dempcy R. O., Browne K. A., Bruice T. C. Synthesis of a thymidyl pentamer of deoxyribonucleic guanidine and binding studies with DNA homopolynucleotides. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6097–6101. doi: 10.1073/pnas.92.13.6097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Don R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. doi: 10.1093/nar/19.14.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hurley L. H., Reynolds V. L., Swenson D. H., Petzold G. L., Scahill T. A. Reaction of the antitumor antibiotic CC-1065 with DNA: structure of a DNA adduct with DNA sequence specificity. Science. 1984 Nov 16;226(4676):843–844. doi: 10.1126/science.6494915. [DOI] [PubMed] [Google Scholar]
  6. Lamm G. M., Blencowe B. J., Sproat B. S., Iribarren A. M., Ryder U., Lamond A. I. Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts. Nucleic Acids Res. 1991 Jun 25;19(12):3193–3198. doi: 10.1093/nar/19.12.3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  8. Lukhtanov E. A., Kutyavin I. V., Gamper H. B., Meyer R. B., Jr Oligodeoxyribonucleotides with conjugated dihydropyrroloindole oligopeptides: preparation and hybridization properties. Bioconjug Chem. 1995 Jul-Aug;6(4):418–426. doi: 10.1021/bc00034a012. [DOI] [PubMed] [Google Scholar]
  9. Monia B. P., Lesnik E. A., Gonzalez C., Lima W. F., McGee D., Guinosso C. J., Kawasaki A. M., Cook P. D., Freier S. M. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem. 1993 Jul 5;268(19):14514–14522. [PubMed] [Google Scholar]
  10. Nielsen P. E., Egholm M., Buchardt O. Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconjug Chem. 1994 Jan-Feb;5(1):3–7. doi: 10.1021/bc00025a001. [DOI] [PubMed] [Google Scholar]
  11. Smits H. L., Tieben L. M., Tjong-A-Hung S. P., Jebbink M. F., Minnaar R. P., Jansen C. L., ter Schegget J. Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types. J Gen Virol. 1992 Dec;73(Pt 12):3263–3268. doi: 10.1099/0022-1317-73-12-3263. [DOI] [PubMed] [Google Scholar]
  12. Wagner R. W., Matteucci M. D., Lewis J. G., Gutierrez A. J., Moulds C., Froehler B. C. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science. 1993 Jun 4;260(5113):1510–1513. doi: 10.1126/science.7684856. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES