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

262 Case-Green, Pritchard, and Southern
flanking region to the repeat is hybridized to an array of oligonucleotides that contain a
guide registration sequence plus a varying number of repeats. The ligase can only
join the labeled oligonucleotide to the oligonucleotides of the array where a perfect
duplex junction is formed. This only occurs for oligonucleotides with repeat length
equal to those of the target (18).
The polymerase method is similar. The STRs must have a base of the repeat unit
that differs from the first base in the flanking sequence following the repeats. Labeled
nucleotide triphosphate complementary to the first base after the repeat and DNA
polymerase is added. The labeled nucleotide is incorporated only where the repeat
number on the array matches that of the target. As confirmation, the reaction can be
performed using a labeled nucleotide triphosphate complementary to the first base of
the repeat. Only array oligonucleotides shorter than the target repeat size are extended
in this case.
1.5. Discussion
Three broad classes of assays useful with oligonucleotide arrays are described
above: allele-specific hybridization; primer extension by polymerase (minisequencing);
and ligase assay. All have been used in solution and adapted to arrays. Each assay
incorporates an initial hybridization of target nucleic acid (usually PCR product) to
the oligonucleotide array.
Allele-specific hybridization and the related technique of sequencing by hybridization
for sequencing and resequencing nucleic acids has been under development and in
use for some time (8,14). Hybridization has several well-documented complications, the
major one being the variability of hybridization yield between different oligonucleotide
probes against the nucleic acid target. This variability has two causes, detailed as
follows:
• Sequence-dependent hybridization efficiency. Where oligonucleotides of the array are
of the same length, then the stability of the duplex formed with the nucleic acid target
will depend on the sequence of the oligonucleotides and the type of target (19). This
effect of base composition in DNA/DNA duplexes is lessened by the use of salts, such as
tetramethylammonium chloride as hybridization buffer (20). The stabilities of each set of
duplexes could be measured by thermal denaturation in solution but this is extremely time
consuming and therefore impractical.
• Differences in accessibility of the target to oligonucleotide probes. Secondary and tertiary
structure in both the target and oligonucleotides can prevent hybridization. The problem
can be alleviated by degrading the target to short fragments.
Selecting a set of oligonucleotide probes that give similar hybridization yields
between loci and similar differences in hybridization yield between the allele specific
oligonucleotides at each locus, under the same set of hybridization conditions, is
difficult. In practice, the problem can be minimized by building redundancy into oligonucleotide
arrays (14). Each locus and each allele is represented by oligonucleotides
with different lengths so that at least one pair of allele-specific oligonucleotides will
exhibit the correct hybridization characteristics.
In enzymatic assays, the hybridization step is used to capture the target. This step
can be done at low stringency, allowing efficient hybridization at all ASOs. Alleles are
then discriminated by either polymerase or ligase, which add a labeled reporter group