Addendum: Mar. 11, 2004

I've decided to stop pursuing a patent for Matrix Sequencing (US appl # 20030036073), and what appears to be a completely novel invention is now completely free to anyone who wishes to perfect and utilize it.

I've had difficulty with the US Patent Office. They have presented three references in the rejection of the invention.

1) Mills, Jr et al 6,083,726;
2) Barany et al 6,534,293 B1; and
3) Weindel et al 6,214,549.

Regardless this is a good time to do what I think I wanted from the beginning to simply protect the idea so as to make it freely accessible to everyone. If you require a written signed statement to this effect please contact me.

There are better things to do than fight this sort of thing. For example if you have not looked at another invention I'm working on entitled "Ligand-Conjugated Polynucleotides and Microarrays of Combinatorial Libraries Thereof" (Provisional Patent filing date 12/30/03) I urge you to do so.

Link to Invention

This invention has vast potential, perhaps surpassing the utility of antibodies. If it continues to be novel, it is likely I will give it away, along with the protein sequencing inventions, after sufficient protection

Final Matrix Sequencing claims rejected by reference to Weindel et al 6,214,549

1) A set of probes utilized together in sequencing a target, each probe being distinct in number or arrangement of universal nucleotides, yet common in having the capacity to specifically and precisely hybridize to the target.

2) The set of probes described by claim 1, further characterized as comprising:

i) first sections (registering sequences) which upon hybridizing to copies of a target, identically position within the target sequence, their junctures with
ii) second sections consisting of universal nucleotides, the number of which is distinct for each probe.

4) The set of probes described by claim 2, wherein each probe is support-affixed.

5) A set of probes utilized together in sequencing a target, said probes comprising:

i) first sections (registering sequences) comprising an identical sequence of base-specific nucleotides, adjoined to
ii) second sections consisting of universal nucleotides, the number of which is distinct for each probe.

7) The set of probes described by claim 5, wherein each probe is support-affixed.

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US Patent Application 20030036073 / Filed May 29, 2002

Summary
A novel method of DNA sequencing utilizing a microarray (biochip) of polynucleotide probes, which unlike Sequencing by Hybridization (SBH), allows serial reading of a target sequence in a fashion similar to electrophoretic gels.

Figure 1 is important in that it describes the idea that led to the conception of the very interesting process described in Figures 2-4.

The sequencing process in Figure 1 advances upon a previously described form of nonelectrophoretic sequencing involving stepwise addition of reversibly 3'-blocked and labeled nucleotides (Cheeseman, P. & Metzker, et al). The process of Figure 1 begins with a matrix support wherein identical 3' reversibly-blocked primers, or primer sets, are arrayed into each section and hybridized to targets. Step #1 is to optically mask out one section and then deblock with light all those those 3' termini not masked. In step #2 the 3' ends of the deblocked primers are extended with reversibly blocked nucleotides (Nts). Subsequent to removing or disabling the noncoupled nucleotides in step #3, a second optical masking, this time involving both the first and second sections, is again followed by deblocking those 3' termini not masked. The process of progressive masking, deblocking, and nucleotide addition is repeated until all the sections have been processed. The final step, not shown, involves deblocking all the 3' termini and extension with distinctly labeled terminating nucleotides.

Note unlike prior stepwise sequencing processes, the use of a microarrayed biochip eliminates the need for reversibly-blocked labeled nucleotides and their cycled addition, detection and delabeling. That is, in the process of Figure 1, multiple initially identical primers, hybridized to identical targets, are differentially extended, and then labeled only once.

An important derivation of the process in Figure 1 involves ligation of oligonucleotides rather then polymerase incorporation of nucleotides (Macevicz, S.C. & Southern, et al).

Envisioning the nested primers resultant from the process in Figure 1, the sequencing process in Figure 2 becomes readily apparent.

"Termed Matrix Sequencing, this process completely obviates the stepwise construction the nested primers as in Figure 1, via the use of prefabricated microarrays of nested universal base-containing primers. Universal bases ("X") are those which match up ("base-pair") with any of the naturally occurring bases (Nichols, et al & Berger, et al). An important feature of Matrix Sequencing primers is the "registering sequence" (herein the underlined M13 universal primer) which perfectly aligns the hybridization of the universal base-containing sections of the primers along the target."

Subsequenct to the hybridization of the ssDNA targets to the registering sequences, minisequencing is effected by primer extension with distinctly labeled "*" chain-terminating nucleotides.

Figure 3 is as Figure 2 except that at the 3' terminal nucleotide of the nested universal base-containing primers is one of the four natural nucleotides which overlaps and interrogates the target nucleotide in question.

Upon addition of polymerase and polymerizable nucleotides, at least some of which are labeled "*", only those primers whose 3' ends are perfectly hybridized are extended and labeled.

Figure 4 exemplifies the oligonucleotide ligation version of Matrix Sequencing.

Figure 5 exemplifies yet another derivation of Matrix Sequencing termed Scanning Mismatch Sequencing.

In this example all the probes have the same length and number of universal nucleotides, yet their arrangement is such as to allow scanning of the target sequence one nucleotide at a time. Loss of terminal labeling of the probes that are cleaved distinguishs matched from mismatched target hybridized probes.

An exceptionally promising alternative to cleaving mismatches is to identify them electronically (Kelly, et al).

Note the potential for multiplex sequencing in the above examples, especially when combined with Sequencing by Hybridization. Alternatively, the registering sequences can be complementary to engineered primer sequences, rather than sequences inherent in the naturally occurring DNA sequences; and that the technology to create the required large number of clones is at hand (Brenner, et al).

References

Brenner, et al (1999) In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs. Proc Natl Acad Sci U S A 1999 97(4):1665

Berger, et al (2000) Universal bases for hybridization, replication and chain termination. Nucleic Acids Res. Aug 1;28(15):2911-2914

Cheeseman, P. (1994) Method for sequencing polynucleotides. United States Patent #5,302,509

Kelly, et al (1999) Single-base mismatch detection based on charge transduction through DNA. Nucleic Acids Res 1999 Dec 15;27(24):4830-7

Macevicz, S.C. (1998) DNA sequencing by parallel oligonucleotide extensions. United States Patent #5,750,341

Metzker, et al (1994) Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates. Nucleic Acids Res. Oct 11;22(20):4259-67

Nichols, et al (2000) A universal nucleoside for use at ambiguous sites in DNA primers. Nature 1994 Jun 9;369(6480):492-3

Southern, E. & Cummins, W.J. (1998) Tag reagent and assay method. United States Patent #5,770,367

Addendum 3/5/06

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence. Twist CR, et al Anal Biochem. 2004 Apr 1;327(1):35-44

Detection of individual oligonucleotide pairing by single-molecule microscopy. Trabesinger W, et al Anal Chem. 1999 Jan 1;71(1):279-83