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. 1991 Nov;44(11):924–927. doi: 10.1136/jcp.44.11.924

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

S F An 1, K A Fleming 1
PMCID: PMC496633  PMID: 1752983

Abstract

A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.

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Selected References

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

  1. Bramwell N. H., Burns B. F. The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. Exp Hematol. 1988 Sep;16(8):730–732. [PubMed] [Google Scholar]
  2. Coates P. J., d'Ardenne A. J., Khan G., Kangro H. O., Slavin G. Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections. J Clin Pathol. 1991 Feb;44(2):115–118. doi: 10.1136/jcp.44.2.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Impraim C. C., Saiki R. K., Erlich H. A., Teplitz R. L. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun. 1987 Feb 13;142(3):710–716. doi: 10.1016/0006-291x(87)91472-0. [DOI] [PubMed] [Google Scholar]
  4. Jackson D. P., Lewis F. A., Taylor G. R., Boylston A. W., Quirke P. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. J Clin Pathol. 1990 Jun;43(6):499–504. doi: 10.1136/jcp.43.6.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lai-Goldman M., Lai E., Grody W. W. Detection of human immunodeficiency virus (HIV) infection in formalin-fixed, paraffin-embedded tissues by DNA amplification. Nucleic Acids Res. 1988 Aug 25;16(16):8191–8191. doi: 10.1093/nar/16.16.8191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Lampertico P., Malter J. S., Colombo M., Gerber M. A. Detection of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver tissue by the polymerase chain reaction. Am J Pathol. 1990 Aug;137(2):253–258. [PMC free article] [PubMed] [Google Scholar]
  7. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  8. Lo Y. M., Mehal W. Z., Fleming K. A. False-positive results and the polymerase chain reaction. Lancet. 1988 Sep 17;2(8612):679–679. doi: 10.1016/s0140-6736(88)90487-4. [DOI] [PubMed] [Google Scholar]
  9. Lo Y. M., Mehal W. Z., Fleming K. A. In vitro amplification of hepatitis B virus sequences from liver tumour DNA and from paraffin wax embedded tissues using the polymerase chain reaction. J Clin Pathol. 1989 Aug;42(8):840–846. doi: 10.1136/jcp.42.8.840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Rogers B. B., Alpert L. C., Hine E. A., Buffone G. J. Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction. Am J Pathol. 1990 Mar;136(3):541–548. [PMC free article] [PubMed] [Google Scholar]
  11. Shibata D., Hu E., Weiss L. M., Brynes R. K., Nathwani B. N. Detection of specific t(14;18) chromosomal translocations in fixed tissues. Hum Pathol. 1990 Feb;21(2):199–203. doi: 10.1016/0046-8177(90)90129-s. [DOI] [PubMed] [Google Scholar]
  12. Tokuda Y., Nakamura T., Satonaka K., Maeda S., Doi K., Baba S., Sugiyama T. Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J Clin Pathol. 1990 Sep;43(9):748–751. doi: 10.1136/jcp.43.9.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wu A. M., Ben-Ezra J., Winberg C., Colombero A. M., Rappaport H. Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffin-embedded specimens. Lab Invest. 1990 Jul;63(1):107–114. [PubMed] [Google Scholar]
  14. de Franchis R., Cross N. C., Foulkes N. S., Cox T. M. A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Res. 1988 Nov 11;16(21):10355–10355. doi: 10.1093/nar/16.21.10355. [DOI] [PMC free article] [PubMed] [Google Scholar]

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