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Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides.
G Caetano-Anollés1, B J Bassam, P M Gresshoff
1Institute of Agriculture, University of Tennesse, Knoxville 37901-1071.
Molecular & General Genetics : MGG
|November 1, 1992
Summary
DNA amplification fingerprinting (DAF) relies on short primers to create DNA fingerprints. The 3' end of the primer, at least 8 bases long, is crucial for effective DNA amplification and fingerprint pattern generation.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- DNA amplification fingerprinting (DAF) uses short oligonucleotide primers for DNA fingerprint generation.
- Understanding primer characteristics is key to optimizing DAF patterns.
Purpose of the Study:
- To investigate how primer sequence and length influence DNA amplification fingerprint patterns.
- To assess the impact of primer-template mismatches on DAF results.
- To define the critical region of primers responsible for DNA amplification.
Main Methods:
- DNA amplification was performed using sequence-related primers of varying lengths.
- Primers were systematically shortened from the 5' terminus to evaluate length effects.
- Single base changes were introduced within the primer sequence to study mismatch effects.
- Amplification was conducted across a range of annealing temperatures (15°C to 70°C).
Main Results:
- Unique DNA fingerprints were generated only with primers 8 nucleotides or shorter.
- Primer length and sequence significantly altered amplification products, particularly within the first 8 bases from the 3' terminus.
- Increasing annealing temperature reduced amplification efficiency for shorter primers without altering the critical 8-base region.
Conclusions:
- A 3'-terminal domain of at least 8 nucleotides largely dictates DNA amplification in DAF.
- Sequences beyond this domain modulate amplification, and not all template annealing sites are efficiently amplified.
- A model suggests competition between primer annealing and template hairpin structures influences product amplification.