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PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis.

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis.

Authors+Show Affiliations

Krishna Institute of Medical Sciences (KIMS) Foundation and Research Centre, KIMS Hospitals, Hyderabad, India. Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology (VIT), Vellore, India.Krishna Institute of Medical Sciences (KIMS) Foundation and Research Centre, KIMS Hospitals, Hyderabad, India. Department of Genetics, Osmania University, Hyderabad, India.Diagnostics Division, KIMS, Hyderabad, India.Diagnostics Division, KIMS, Hyderabad, India.Krishna Institute of Medical Sciences (KIMS) Foundation and Research Centre, KIMS Hospitals, Hyderabad, India.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31695579

Citation

Panchal, Nagesh Kishan, et al. "PCR Amplifiable DNA From Breast Disease FFPE Section for Mutational Analysis." Journal of Biomolecular Techniques : JBT, 2019.
Panchal NK, Bhale A, Chowdary R, et al. PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis. J Biomol Tech. 2019.
Panchal, N. K., Bhale, A., Chowdary, R., Verma, V. K., & Beevi, S. S. (2019). PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis. Journal of Biomolecular Techniques : JBT, doi:10.7171/jbt.20-3101-001.
Panchal NK, et al. PCR Amplifiable DNA From Breast Disease FFPE Section for Mutational Analysis. J Biomol Tech. 2019 Oct 31; PubMed PMID: 31695579.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - PCR Amplifiable DNA from Breast Disease FFPE Section for Mutational Analysis. AU - Panchal,Nagesh Kishan, AU - Bhale,Aishwarya, AU - Chowdary,Radhika, AU - Verma,Vinod Kumar, AU - Beevi,Syed Sultan, Y1 - 2019/10/31/ PY - 2019/11/8/entrez PY - 2019/11/7/pubmed PY - 2019/11/7/medline KW - DNA extraction KW - DNA fragmentation KW - deparaffinization KW - lysis buffer KW - salt treatment JF - Journal of biomolecular techniques : JBT JO - J Biomol Tech N2 - Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis. SN - 1943-4731 UR - https://www.unboundmedicine.com/medline/citation/31695579/PCR_Amplifiable_DNA_from_Breast_Disease_FFPE_Section_for_Mutational_Analysis L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31695579/ DB - PRIME DP - Unbound Medicine ER -