Optimization of PCR amplification and sequencing of GC-rich protein-coding region of CEBPA (CCAAT/enhancer binding protein alpha) in acute myeloid leukemia patients using betaine
Document Type
Article
Department
Biological and Biomedical Sciences; Centre for Regenerative Medicine and Stem Cell Research; Haematology/Oncology
Abstract
Background: CEBPA, a GC rich gene encodes for transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) which is essential for granulocyte development. Mutation(s) in CEBPA leads to block in myeloid cells differentiation which is characteristic of acute myeloid leukemia (AML). In addition, mutation(s) in CEBPA has been associated with prognosis in AML. Therefore, analysis of protein-coding sequence of CEBPA is critical in patients diagnosed with AML. This study aimed to optimize PCR amplification followed by Sanger sequencing of GC rich regions of CEBPA in AML patients using betaine which is a known PCR additive with relatively less inhibitory effect.
Methods and results: PCR amplification using two sets of primer was performed with varying concentrations of betaine and MgCl2 at different annealing temperatures. Purified amplified PCR products were used for Sanger sequencing. PCR amplification of both 576 bp and 703 bp specific amplicons was achieved consistently with 1 M betaine and 2mM MgCl2. We found that increase in betaine inhibits PCR at higher annealing temperature which can be attributed to its ability to decrease the melting temperature of the DNA template. The optimized protocol did not interfere with the downstream applications such as PCR-product purification and Sanger sequencing in AML patients.
Conclusion: These findings establish a potentially cost-effective and feasible method for sequencing protein-coding region of CEBPA in AML patients.
Publication (Name of Journal)
Molecular biology reports
DOI
10.1007/s11033-026-12388-4
Recommended Citation
Bilwani, F.,
Iqbal, F.,
Khurshid, M.
(2026). Optimization of PCR amplification and sequencing of GC-rich protein-coding region of CEBPA (CCAAT/enhancer binding protein alpha) in acute myeloid leukemia patients using betaine. Molecular biology reports, 53(1), 1-9.
Available at:
https://ecommons.aku.edu/pakistan_fhs_mc_bbs/1130