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Annex 2. Analysis of documents cited in the PCT research report

Sections taken from documents D1-D7 related to our patent 2007-2008 are written in italics and we have underlined the critical parts of any claims or arguments to emphasize the differences with our PCT application.

D1 WO 00/43538 (Université de Montréal); 27 July 2000

D2 (Biotechniques, 2002, Brukner et al).

1. 
   In the body of the text and in the claims of D1, there is a requirement for selection of probes (OL) under stringent conditions, in order to minimize mismatches. In our present PCR/CA2007/001398 it is exactly the opposite—in order to get better discriminatory probes (recognizing only intended targets), selection is done under non-stringent conditions to maximize mismatches.
   

D1: WHAT IS CLAIMED IS 1. A process for generating an oligonucleotide library which originates from a chosen biological material, comprising: a) generating random oligonucleotides, wherein said oligonucleotides are of a uniform length comprising a central segment of randomly varied bases and segments of a defined sequence flanking the central segment on each side; b) hybridizing said random oligonucleotide mixture of a) with a nucleic acid template of biological origin under hybridization conditions which enable the formation of duplexes, while minimizing or abrogating mismatches; c) separating said duplexes from non-duplexed material; and d) amplifying said hybridized oligonucleotides…

2. 
   The same procedure of selection is presented in D2 (Biotechniques, 2002, Brukner et al). However, during the writing of MS it was realized and discussed (by citing literature) that existence of mismatches was not detrimental to the generation of diagnostics probes (OL). Still, the procedure was following stringent conditions of selection. The crucial effect of selection under non-denaturing (non-stringent conditions) was not realized in D1 and D2.
   
3. 
   Negative selection-subtraction was completely changed in our PST application, compared with documents D1 and D2, and accommodated to make simultaneous positive and negative selections in the second segment of selection cycles. Both D1 and D2 are describing separate (both physically and in terms of timing) positive and negative selection (subtraction). Simultaneous selection (positive, forward and negative-subtractive) is an essential feature not because it is faster, but because it will guarantee affinity distribution of selected probes, which will be higher for intended targets, but lower for unintended targets. Only affinities falling in this range will have differential binding performance.
   

D3 US 2005/0175989 Claims in this patent include particular HPV hybridization probes. However, claim 15 (cited here for illustration) is, “A probe which hybridizes to nucleic acid from an HPV subtype, said probe being selected from the group consisting of: SEQ ID NO: 1-SEQ ID NO: 12 and sequences fully complementary thereto.” Our PCT application is describing hybridization probes that do not rely only on exact and/or full complement of targets. In fact, this is the essential feature of our probes. Further, our method does not even need knowledge of nucleic acid target sequences, just the presence of targets and non-targets. However, for the purpose of proving the concept and for the purpose of facilitating and controlling the selection (generation of probes) we used publicly available sequence information and chemically synthesized all relevant targets.
D4 WO 99/14377 Present consensus of the majority of the scientific community is that the generation of a spectrum of specific probe sequences that will discriminate plurality of similar targets is not known in the art, which is well described in D7 (our Nucleic Acids Research, 2007 paper) and recent literature related to the hybridization theory. However, D4 offers an example of type-specific probe sequences that do not cover a full spectrum of HPV types and that do not have discrimination power over a full set of HPV types.

Description:

“Type-specific probe sequences are well known in the art, and any such suitable sequences can be used in the present invention. In the case of HPV, for example, suitable type-specific probes are disclosed in W09914377. The invention is not restricted to the nature or origin of the type-specific probes that are used in hybridization step in the present invention.”

“Claims 1: A process for identification of type-specific polynucleotide sequences in a sample, the process comprising the steps of: (i) contacting polynucleotides from the sample, or derived from the sample, with a plurality of type-specific probes in the context of a solid support, and detection of any type-specific hybridization; and (ii) contacting polynucleotides in the sample, or derived from the sample, with type-specific primers for those types capable of being detected by the hybridization step in step 1, but not so detected, in a type-specific amplification reaction.”

D5 US 2005/0244851 (Affymetrix, INC).

This document describes the use of oligonucelotide probes for the purpose of expression analysis and splicing analysis. However, this document is underestimating the issue of probe specificity, especially differential detection of similar targets. The accumulation of expression-relevant data suggests that the basic premise of nucleic acid hybridizations are not well incorporated into the probe design criterion, leading to the inability to uniquely interpret hybridization signals of multiplex probes with complex nucleic acid samples, like total RNA or something similar. We don’t find any overlap with any segment of our PCT amplification.

Claims (underlined by us)

10. A nucleic acid array comprising a plurality of at least 100,000 probe sets wherein each probe set comprises: a plurality of different perfect match probes, wherein the probes of each probe set are complementary to a single probe selection region, and wherein each probe selection region is a single exon or a subsequence of an exon.
D6 is the sequence of HPV 16 late major capsid protein AF084952, Wang et al, 1998. This document is not relevant since the process of generation/selection of probes:

1. 
   does not require any a priori knowledge of sequence data;
   
2. 
   Our SEQ ID NO: 116 probe is not 100% identical to HPV 16, but contain extra nucleotides both on the 5’ and 3’ sites from the central core of 14 nucleotides full-match containing segments. If an examiner is arguing that the short segment of 14 nucleotide long motif was known in advance, then any single nucleotide, or dinucleotide, or trinucleotide…from any probe is 100% complementary with any target. This raises questions of minimal probe length and of minimal length of a target sequence motif good enough to keep specificity among similar targets…questions that are not theoretically and practically resolved. Any of our probes have short segments of complementarities. To our knowledge, there is no software, algorithm, or known procedure that can pick-up or select very short segments of fully-complementary sequences and can generate the probes that keep differential discrimination among all spectrum of similar targets (HPV type).
   

D7 is our 2007 NAR paper. It is fully relevant to our PCT application, but it does not list all HPV-relevant typing probes due to the fact that the choice of target segment was not optimal (not variable enough). Importantly, this paper was published after we filed the provisional patent application (priority date), so it can not affect novelty.