Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome.
Despite its widespread use, the molecular basis of random amplification is poorly understood. Here the basis of random amplification has been investigated by cloning and sequencing the products of a random amplification of polymorphic DNA (RAPD) amplification from Saccharomyces cerevisiae DNA. The genomic origin of the amplified products was determined by sequence comparison with the S. cerevisiae Genome Database (SGD). This allowed analysis of the degree of identity between the random primer and the primer binding sites on the genome. There was no relationship between RAPD size, GC content and relative abundance. The degree of matching between the primer and the primer binding sites increased towards the 3; end of the primer and decreased towards the 5; end. The maximum number of mismatches observed between primer and primer binding sites was never more than one between positions 1-7 of the primer. Nucleotide compositional biases were also observed upstream and downstream of the primer binding site with a marked preference for AT richness upstream of the primer binding sites and for a GC preference directly following the 3; end of the primer. These findings have important ramifications for primer design for multiplex, low stringency and degenerate polymerase chain reaction (PCR).
McGrath
A
A
Department of Biochemistry, University College Cork, Cork, Ireland.
Higgins
D G
DG
McCarthy
T V
TV
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019105:Random Amplified Polymorphic DNA Technique; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004271:DNA, Fungal