John M. S. Bartlett.pdf - Bio-Nica.info

Ligase Chain Reaction 137
The probe candidates must be tested for sensitivity by determining the lower level of
detection of samples containing organisms of interest or their DNA. Specificity must
be determined by testing related organisms, as well as other organisms likely to be
found in specimens to be tested.
1.3. Target Sequence Selection
The sequence should be unique to the organism to be detected and multiple copies
per organism increases sensitivity. If the ligase reaction is to be used to discriminate
between single base pairs, the ultimate 3′ base is most sensitive, whereas the 5′ base
is also relatively sensitive. Allele-specific PCRs used to detect single nucleotide
polymorphisms are often not discriminating enough to differentiate between SNPs,
whereas the ligase reactions are more discriminating against mismatches, especially
on either side of the ligation site (1). All of the possible mismatches are discriminated
against, but G-T and T-T less well, 1.5% as efficient as the matching nucleotide, while
other mismatches were 80%. When the mismatch was
on the 5′ side the previous two and A-C, A-A, C-A, G-A and T-T, all were all less
discriminated against than when on the 3′ side, same patterns as were found with T4
ligase. Intentionally introducing a mismatch in the third site from the 3′ end of the
probes increased the discriminating power. Nucleotide analogs in the probes in the
2 and 3 location from the 3′ end also increased discriminatory power. Site-directed
mutagenesis was used on Tth and mutants that increased discriminating power 4- and
11-fold were found by Luo et al. (3).
1.4. Detection of Amplification Products
Multiplex LCR using a mixture of probes differing by the 3′ nucleotide involved
in the ligation, that are labeled by being one or two extra bases on the nonligating
5′ end, allows polyacrylamide gel electrophoresis (PAGE) differentiation of the one
or two base changes by differences in migration in PAGE (1). The probes can also be
labeled with several different, easily detected labels such as: 32P, fluorescent labels,
immunologically detectable haptens (digoxigenen), (Roche Molecular Diagnostics,
Indianapolis, IN) and after amplification and electrophoresis the signal is detected
by Southern blot to determine if ligation has taken place. Ligated products can also
be detected by having immuno-capture of one of the probe ends and after washing,
detecting the second probe with an enzyme conjugate. Only ligated product will be
captured and also have the end with the ligand for which the enzyme conjugated
antibody is specific, IMx (Abbott) utilizes this method. The latter method is also
employed in the commercially sold Abbott LCx for C. trachomatis and N. gonorrhoeae.
The comparison of eight different nonradioactive methods of detecting the LCR
products was reported by Winn-Deen (19).
1.5. Contamination Control
One problem with LCR is that the target is amplified, resulting in a contamination
risk. The method commonly used to inactivate PCR products does not work because of