Hybridisation Portion Control (HPC™) Technology

The main principle of the oligonucleotide applied to ACP™ Technology can be extended to the field of nucleic acid Hybridisation as the name Hybridisation Portion Control oligonucleotide (HPC™ oligonucleotide). The HPC™ oligonucleotide insures a very specific Hybridisation reaction to a target nucleotide sequence, such that a variety of analyses using Hybridisation can be performed with higher reliability.

The HPC™ Technology is directed to the HPC™ oligonucleotide with its novel structure having (i) a first Hybridisation portion having a nucleotide sequence substantially complementary to a target nucleotide sequence, (ii) a second Hybridisation portion having a pre-selected arbitrary nucleotide sequence, and (iii) a regulator portion bridging the first Hybridisation portion and the second Hybridisation portion which plays a key role in Hybridisation reaction by its effect, or performance, on the first and second Hybridisation portions.

The HPC™ oligonucleotide utilises two independent Hybridisation steps which require two different Hybridisation temperatures. During the two independent hybridisations, the 3'-end portion is used to form a hybrid with the template at the first Hybridisation steps and the 5'-end portion is used to form a hybrid with its complementary sequence at the second Hybridisation step. The second Hybridisation step evaluates the quality of spotting and immobilisation of HPC oligonucleotide. During the two individual hybridisations, the first and second hybridisation portions work independently due to the effect of the regulator in the HPC oligonucleotide as described in the following 1~5 steps.

Step 1: The regulator is capable of restricting a Hybridisation portion of the oligonucleotide to the first Hybridisation portion (3'-end portion) and excludes the second Hybridisation portion (5'-end portion) at the first Hybridisation step. Therefore, the Hybridisation sequence of the oligonucleotide can be precisely controlled, which makes it possible to design an oligonucleotide capable of having a desired number of Hybridisation sequences. This is particularly useful when a Hybridisation portion of an oligonucleotide has to be limited (e.g., single nucleotide polymorphism (SNP) genotyping, DNA microarray screening and detection of differentially expressed genes).

Step 2: The second Hybridisation portion not complementary to a target nucleotide sequence leaves the first Hybridisation portion free to hybridise with its target nucleotide sequence when the HPC™ oligonucleotide is bound to a substrate such as nylon membrane or glass, thereby increasing Hybridisation strength (efficiency) of the first Hybridisation portion.

Step 3: The increased Hybridisation strength (efficiency) of the first Hybridisation portion allows Hybridisation reaction to be performed under highly stringent conditions which include higher Hybridisation and washing temperatures, so that the Hybridisation specificity of the first Hybridisation portion is increased.

Step 4: The above-mentioned features of the present HPC™ oligonucleotide leads to the dramatic enhancement of the Hybridisation specificity so that even one mismatch throughout the hybridised duplex may be discriminated from a complete match: Thus, the HPC™ oligonucleotide is particularly useful for the identification of a nucleotide variation in a target nucleic acid, for example, single nucleotide polymorphisms and point mutations: HPC also provides an oligonucleotide with a high tolerance in "parameters" for probe design such as oligonucleotide length, Hybridisation temperature and GC content.

Step 5: The second Hybridisation portion also permits the verification of the first Hybridisation results, which can exclude the first Hybridisation data from erroneous results due to artificial effects such as the failures of immobilisation of oligonucleotide on substrate and establishment of optimal Hybridisation conditions.

Fig. 1. SNP genotyping of human p53 gene by allele-specific dot blot Hybridisation using three different types of fragments amplified from homozygous wild-type, homozygous mutant-type and hetero-type genomic DNA. Wild, mutant and hetero type fragments were respectively used as probes at first Hybridisation The positive control oligo was used as a probe at second Hybridisation W: wild-type oligo, M: mutant-type oligo.