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Amplification of intermediate-size DNA sequences from formalin and B-5 fixed tissue by polymerase chain reaction

D Crisan1, J C Mattson

  • 1Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI 48073.

Clinical Biochemistry
|April 1, 1992
PubMed

Insights

DNA analysis of fixed bone marrow biopsies is feasible using polymerase chain reaction (PCR) amplification. Formalin fixation preserves DNA integrity better than B-5 fixation, enabling successful PCR amplification and sickle cell anemia genotyping.

Area of Science:

  • Molecular Biology
  • Genetics
  • Histopathology

Background:

  • Retrospective analysis of DNA from fixed bone marrow biopsies is challenging due to DNA degradation.
  • Conventional DNA analysis methods may fail with degraded DNA from paraffin-embedded specimens.

Purpose of the Study:

  • To evaluate the feasibility of DNA analysis from formalin-fixed bone marrow specimens using PCR.
  • To compare DNA quality and PCR success rates between formalin and B-5 fixation methods.
  • To assess the utility of PCR amplification and Cvn I digestion for sickle cell mutation detection.

Main Methods:

  • DNA extraction from formalin-fixed and B-5-fixed bone marrow biopsy specimens.
  • Polymerase chain reaction (PCR) amplification of a 725 bp beta-globin gene sequence.
  • Allele-specific digestion of amplified products using Cvn I enzyme.
  • Genotyping for beta A/beta S alleles to detect sickle cell mutations.

Main Results:

  • DNA from B-5-fixed specimens showed more degradation than from formalin-fixed specimens.
  • PCR amplification of the target beta-globin sequence was successful only with DNA from formalin-fixed specimens.
  • Accurate genotyping for sickle cell-related genotypes (beta A/beta A, beta A/beta S, beta S/beta S) was achieved using the amplified products.

Conclusions:

  • Intermediate-size DNA sequences can be successfully amplified and analyzed from formalin-fixed bone marrow specimens.
  • Formalin fixation is a superior method for preserving DNA integrity in bone marrow biopsies for subsequent PCR-based genetic analysis compared to B-5 fixation.
  • PCR-based genotyping is a viable method for retrospective analysis of genetic mutations in fixed bone marrow samples.

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