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

258 Case-Green, Pritchard, and Southern
reagent use and allows the array to be cut into strips in a direction perpendicular to
the array synthesis.
Alternatively, a sequence that flanks the variable base is synthesized; the cell is
displaced by half a cell’s width and a base corresponding to one of the alleles is added,
the cell is then moved to cover the other half and the other varying base added. The
cell can then be returned to its original position and any further bases common to both
allele specific probe oligonucleotides added.
1.1.2. STR Array Fabrication
The basic requirements for an array suitable for use in STR typing are shown in
Fig. 3. All the oligonucleotides of the array include a sequence complementary to the
region immediately adjacent to the repeats. This registration sequence forms duplex
between the target and the array oligonucleotides and aligns the start of the repeat
of the oligonucleotides with that in the target. The array oligonucleotides vary in the
number of repeat units that they contain on top of the flanking registration sequence.
Synthesis of these arrays can be carried out in a combinatorial manner. Figure 4 shows
a scheme for the synthesis of an array for typing the FES locus (10).
1.2. Preparation of Target DNA
To achieve good hybridization yields and allow efficient extension of the array
oligonucleotides, a single-stranded target is required. Depending on the type of assay
the target may be either labeled or unlabeled. Because the DNA for analysis is usually
genomically derived material, an amplification step is normally performed.
For hybridization assays, the most common target species is body labeled RNA,
prepared by carrying out a polymerase chain reaction (PCR) of the required region
using a primer that includes an RNA polymerase promoter sequence and transcribing
the product with the appropriate enzyme. The reaction mixture also contains labeled
nucleotide triphosphate.
For assays involving enzymes, a single-stranded unlabeled DNA target is required.
A two-step procedure can be used where the sample is first amplified using the PCR
and the unwanted strand digested away using an exonuclease (11). The primer for the
strand to be retained is synthesized with approximately the last five internucleotide
linkages at the 5′ end as phosphorothioate (12), which protect the strand against
nuclease degradation. After PCR, T7 gene 6 exonuclease is added to the product to
digest away the unwanted, (unphosphorothioated) strand. The resulting single-stranded
DNA can often be used without further purification but, if required, standard purification
techniques could be used, for example, Sephadex or Qiaquick columns.
1.3. Hybridization Assays
Target alleles are distinguished by their ability to hybridize to complementary allele
specific oligonucleotides (ASOs) on the array (Fig. 5A) (8). Hybridization yield and
discrimination depend on temperature, salt concentration, time, and target concentration.
The sequence of the oligonucleotide also affects both yield and discrimination.
Many alleles can be simultaneously examined on the same array, but this requires careful
choice of both oligonucleotide sequence and hybridization and washing conditions
to achieve the maximum discrimination under one set of conditions. Computer-based