Advanced Techniques in Diagnostic Microbiology

TABLE 19.1. Comparison of inactivation protocols used in nucleic acid tests.
Method Application Conditions Advantages Disadvantages References
UV light
irradiation
Preamplification Lower G + C content,
>500 bp amplicon
UNG Preamplification Lower G + C content,
>100 bp amplicon
Photochemical
cross-linkers
Postamplification Lower G + C content,
>100 bp amplicon
Primer hydrolysis Postamplification No specific
requirements
Hydroxylamine
treatment
Restriction
endonuclease
Postamplification Higher G + C content,
>100 bp amplicon
Preamplification No specific
requirements
Inexpensive. Simple
procedure. No
changes to protocol.
Efficacy varies Sakar and Sommers, 1990;
Ou et al., 1991; Belak and
Ballagi-Pordany, 1993
Simple procedure Changes necessary in amplification
cycling and master mix. More
expensive than other methods.
Simple procedure Additional instruments required.
Can change molecular mass of
products, which affects
electrophoresis.
No effect on amplicon
analysis
Effective on some
amplicons
Effective on some
amplicons
Necessary for reagent addition
postamplification. Efficacy varies.
Necessary for reagent addition
postamplification. Some
amplicon changes may affect
analysis. Hydroxylamine is a
mutagenic agent.
Espy et al., 1993; Rys and
Persing, 1993
Cimino et al., 1993; Espy
et al., 1993; Rys and
Persing, 1993
Rys and Persing, 1993
Aslanzadeh, 1993
May lengthen processing time. DeFilippes, 1991
UNG, uracil-N-glycosylase.
317