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

136 Benjamin, Smith, and Waites
amplification occurs. The two complementary pairs can also, at low frequency, be blunt
end ligated to each other and serve as template for amplification even though no target
sequence was present in the original sample. Early work showed at least 10-fold greater
efficiency of ligation of perfectly matched compared to a mismatched nucleotide at
the ligation junction (1,8,12).
LCR-based systems have some advantages over the PCR-based amplification systems.
Because there is no newly synthesized DNA, misincorporated nucleotides are not
replicated in the product allowing amplification of a different sequence than that found
in the target nucleic acid. The LCR reactions are also more specific for the 3′ nucleotide
allowing for higher discriminatory power against mismatches at a single chosen site (1,3).
Thus, LCR is very useful for determining the nucleotide at a specific site such as a single
base change, e.g., single-nucleotide polymorphisms (SNPs) used in mapping complex
genomes. The LCR cycle has only two short steps allowing for shorter amplification
times. The usually small target of LCR, 36 to 60 nucleotides, does not require highquality
large fragment nucleic acids (13). The commercial LCR kit, the Abbott LCx
System (Abbott Diagnostics, Abbott Park, IL, USA) is less affected by inhibitors in some
specimens, such as fresh urines compared to PCR (14). However, the LCR is still subject
to contamination that is at least as much of a problem as for the PCR.
There are several modifications to the simple LCR that have been used to overcome
some of the problems that are inherent in this method. Owing to the specificity of the
ligase reaction for perfect matches that is complicated by the blunt end ligation of
double stranded probes PCR, the Ligase Detection Reaction (LDR) was developed
(15). The amplification step for LDR is PCR using outside primers, then only one
pair of adjacent primers is used to detect the proper sequence in the PCR product
(1,7). Because there are no double stranded oligonucleotides to blunt end ligate, the
background is very low and the linear detection of the PCR amplified sequence can
be detected using ligation of oligonucleotides. Gap LCR utilizes a few base pair gaps
between the two pair of oliogonucleotides on the primer, thus requiring a thermostable
polymerase (Taq polymerase) to fill in the gap before ligation of adjacent primers can
occur. The Gap LCR prevents amplification of the blunt end ligated primers because the
oligonucleotide pairs cannot be amplified on blunt end ligated products (16,17).
1.1. Ligase
There are four thermostable ligases that can be used in LCR reactions. Taq ligase
was the first described and is commercially available from New England Biolabs
(www.uk.neb.com/neb/index.html) (Beverly, MA). Stratagene (www.stratagene.
com) (La Jolla, CA) produces Pfu which is advertised as having higher ligation
specificity and lower background than Tth that is marketed by Abbott Diagnostics
(www.abbottdiagnostics.com) (Abbott Park, IL) and is used in their LCx diagnostic kit.
Amplicase is sold by Epicenter Technologies (www.epicentre.com) (Madison, WI).
1.2. Probe Design
The most successful probes are 18 to 30 bases in length. Probes with high GC
content or those that form stable secondary structures or dimers should be avoided
if possible. It is difficult to predict activity, so empiric testing is still necessary (18).
The 5′ nucleotide of the adjacent end must be phosphorylated to promote ligation.