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Development and validation of a T7 based linear amplification for genomic DNA
Chih Long Liu1, Stuart L Schreiber, Bradley E Bernstein
1Department of Chemistry and Chemical Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. clliu@fas.harvard.edu
BMC Genomics
|May 13, 2003
Summary
We developed a linear DNA amplification method to reduce bias in genome mapping. This technique, unlike PCR, preserves DNA size distribution and representation for more accurate analysis.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Transcription factor binding and histone modification studies require amplified genomic DNA.
- Current PCR-based amplification methods introduce bias, affecting data accuracy.
- Linear amplification, successful for mRNA, has not been widely applied to genomic DNA.
Purpose of the Study:
- To develop a novel linear amplification protocol for genomic DNA.
- To overcome limitations of PCR-based amplification in genome mapping.
- To reduce amplification bias in genomic DNA analysis.
Main Methods:
- Developed a T7-based linear amplification protocol using terminal transferase to add polyT tails.
- Ensured tail length uniformity with limited ddCTP.
- Generated double-stranded DNA for in vitro transcription (IVT) using a T7-polyA primer adapter.
Main Results:
- Successfully amplified as little as 2.5 ng of genomic DNA.
- Preserved the size distribution of the original DNA fragments.
- Demonstrated reduced bias and improved representation compared to PCR amplification.
- Maintained dynamic range more effectively than PCR.
Conclusions:
- Presented a T7-based linear amplification protocol for genomic DNA.
- The protocol effectively reduces amplification bias in genome mapping.
- This method offers a more accurate alternative to PCR for genomic DNA analysis.