Advanced Techniques in Diagnostic Microbiology

19. Amplification Product Inactivation 313
FIGURE 19.6. Biochemistry of Psoralens. Isopsoralen has been included in the master
mix along with the DNA template when amplification takes place. Postamplification, the
amplicons are treated with UV light to activate isopsoralen. Adducts are created at pyrimidine
residues. Trace contamination of amplicon occurs in the next sample. These treated
amplicons are unable to be amplified by Taq polymerase due to the isopsoralen adducts.
guanine. The resulting compound can bind with adenine and causes its replacement
with thymine if DNA synthesis occurs after treatment (Aslanzadeh, 1993).
This method has been found to work well with smaller amplicons (close to 100 bp)
but is somewhat dependent on G+C concentration. Also, the treatment increases
the molecular weight of the amplicon, which will affect its migration on gels (Rys
and Persing, 1993). A major drawback of this method is that the reaction tube
needs to be opened in order to add hydroxylamine. This can introduce aerosols
that would not have been introduced if some other method was used for product inactivation.
This method has been successfully used with protocols detecting DNA
from HSV and B. burgdorferi (Aslanzadeh, 1993).