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Related Experiment Videos

Effect of highly fragmented DNA on PCR

E M Golenberg1, A Bickel, P Weihs

  • 1Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA. egolenb@biology.biosci.wayne.edu

Nucleic Acids Research
|December 15, 1996
PubMed
Summary

Polymerase chain reactions (PCR) can amplify large fragments from degraded DNA by pre-treating the template. However, this reconstruction process can compete with amplification, reducing product yield.

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Area of Science:

  • Molecular Biology
  • Biochemistry

Background:

  • Degraded DNA presents challenges for accurate amplification in molecular biology techniques.
  • Understanding DNA repair and reconstruction mechanisms is crucial for optimizing PCR.

Purpose of the Study:

  • To characterize polymerase chain reactions (PCR) using degraded DNA.
  • To investigate methods for amplifying large fragments from fragmented DNA templates.
  • To analyze nucleotide incorporation during PCR with degraded DNA.

Main Methods:

  • Pretreatment of degraded genomic DNA by polymerization without primers.
  • Measurement of deoxynucleotide triphosphate (dNTP) incorporation.
  • Comparative PCR experiments with and without degraded DNA addition.
  • Analysis of Taq polymerase activity on DNA reconstruction.

Main Results:

  • Amplification of fragments larger than initial template fragments is possible with overlapping fragment annealing and extension.
  • Nucleotide uptake increases with DNA fragmentation up to a point, then declines with severe degradation.
  • Degraded DNA addition to PCR reduced resultant product, indicating competition between reconstruction and amplification.

Conclusions:

  • Reconstructive polymerization can enable amplification of large fragments from degraded DNA but may compete with amplification.
  • Modified PCR techniques separating reconstructive and amplification steps are suggested to overcome limitations.
  • Taq polymerase's terminal deoxynucleotidyl transferase activity may hinder template reconstruction.

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