Patent Application: US-15614402-A

Abstract:
disclosed herein are materials and processes for a novel method of polynucleotide sequence analysis termed matrix sequencing . the invention utilizes a set of distinct probes , each distinct probe comprising a common first section which specifically hybridizes to a target , and an adjoining second section consisting of universal nucleotides the number of which is distinct for each distinct probe . microarrays of these novel probes , unlike those used in sequencing by hybridization , allow serial reading of the target sequence in a fashion similar to electrophoretic gels .

Description:
a “ nucleotide ” denotes a polynucleotide monomer which resides in , or has the potential to reside in a polynucleotide . there are a myriad of known and synthetically feasible nucleotide derivations . a “ universal nucleotide ” can match up (“ base - pair ”) with the naturally occurring nucleotides with similar tenacity ( 1 - 13 ). a “ degenerate nucleotide ” can base - pair with multiple but not all of the four naturally occurring nucleotide groups ( adenosines , guanosines , cytidines , or thymidines / uridines ). a “ base - specific nucleotide ” can efficiently base - pair to oily one of the four naturally occurring nucleotide groups . a “ probe ” comprises a polynucleotide . in certain processes the probe functions as a primer . probes are preferably covalently or noncovalently affixed , via their 5 ′ or 3 ′ termini , to a support ( s ) prior to or after target hybridization . supports can be of various configurations , composed of various materials , and include soluble polyvalent polymers . preferably the support is a chip wherein distinct probes are arrayed at unique locations ( 14 - 20 ). coded beads are also applicable ( 21 - 24 ). an entity is considered “ distinct ” when in some intrinsic characteristic it is different from others . an unqualified statement such as “ probes ” or “ targets ” optionally indicates multiple identical or distinct entities . as exemplified in fig1 the novel probes of the present invention comprise two adjoining sections . the first probe section termed “ registering sequence ” ( herein the m13 universal primer ) is proximal the support and specifically hybridizes to the target . each distinct probe is affixed to the support at a unique position , and in reality there are many identical probes at each position . registering sequences are preferably 4 or more nucleotides in length . the lengths of the universal nucleotide - containing second sections are limited only by their ability to appropriately hybridize to the targets . note the potential for multiplex sequencing of distinct targets , wherein multiple probe sets having distinct registering sequences are simultaneously utilized . in the first step of fig1 the registering sequences are specifically hybridized to the targets so as to precisely align the hybridization of the incrementally increasing universal nucleotide (“ x ”)- containing second sections . of course the probe composition and the hybridization conditions should be such that probe - target hybridizations are as required . nucleotide derivations can profoundly affect the specificity and efficiency of hybridizations . also , diverse reagents and various proteins may aid in achieving precise probe - target hybridizations ( 26 - 36 ). numerous computer programs and schemes for selection of optimal hybridizing sequences are available ( 37 - 40 ). potentially problematic are unintentional hybridizations by the universal nucleotide - containing second sections ( 8 , 41 ), and preferably these sections hybridize with less stringency than the registering sequences . conditions could even be devised whereby hybridization of probes to targets is accomplished in two stages ; a first stringent stage where only the registering sequences hybridize , followed by the lowering of stringency to allow hybridization by the universal nucleotide - containing second sections . one interesting means by which this might be accomplished is by controlling hybridizations electronically ( 42 - 44 ). conceivably the probes and targets could be designed so that if a probe is not appropriately hybridized to a target , it can be disabled in its capacity to be labeled , such as by enzymatic hydrolysis . continuing with fig1 subsequent to precise hybridization of probe to target , the probe is extended by one fluorescently labeled (“*”) chain terminating nucleotide , the identity of which is specified by the target sequence ( 45 - 53 ). it is of course important that the particular reaction conditions , polymerase , and terminating nucleotides utilized are such that the presence of the universal nucleotides does not preclude extension ( 1 - 2 , 54 , 55 ). a large number of other labeling and detection schemes are applicable . particularly , electronic biochips for detection are attracting considerable attention ( 56 - 63 ). the derivation exemplified in fig2 is similar to that in fig1 except that each probe has one base - specific nucleotide at their 3 ′ end which interrogates a specific target nucleotide . unlike fig1 each target nucleotide being identified requires a subset of four probes rather than one . the probe of each subset that this interrogating nucleotide correctly base - pairs with the target is selectively extended by polymerase incorporation of a labeled nucleotide . conceivably , the probes in this example could have 2 or even 3 terminal base - specific nucleotides interrogating the target sequence . note the redundancy of sequence information due to the probes identifying overlapping dinucleotides ; and the potential to increase the incremental steps from 1 to 2 universal nucleotides . an alternative to the process in fig2 would be to initially have each probe &# 39 ; s 3 ′ terminal nucleotide labeled . after target hybridization , those labeled terminal nucleotides which are mismatched could be selectively removed , such as by an error correcting polymerase . another alternative to fig2 is shown in fig3 . in this derivation termed scanning mismatch sequencing the the probes are equivalent in length due to additional universal nucleotides following the interrogating nucleotide . this may aid in more uniform probe - target hybridizations , and expands the potentially useful labeling and detection schemes . in this example mismatched probes are detected by their selective cleavage and concurrent loss of prelabeled 3 ′ ends ( 64 - 70 ). [ 0034 ] fig4 exemplifies a notably distinct derivation , wherein the hybridized probes are ligated to labeled oligonucleotides as directed by the target . most importantly , the incremental increases in the lengths of the universal nucleotide - containing second sections of the probes can be more than 1 nucleotide ; thus offering the possibility of considerably reducing the number of distinct probes required to sequence a given target . also note ( as shown ) if the incremental increases are smaller than the length of the ligated oligonucleotides , then there is an overlap of sequences read and thus greater accuracy . as in prior figures , it may be advantageous to use subsets of probes which have 1 - 3 base - specific and / or degenerate nucleotides at their distal termini . the termini of the labeled oligonucleotides not intended to be ligated to the probes may be such as to prevent multiple ligations of adjoining ( stacked ) oligonucleotides ( 71 , 72 ). ligation is preferably achieved enzymatically , yet it can also be achieved chemically or by radiation . labeling the required large number of distinct oligonucleotides is preferably via mass spectrometry labels ( 73 ). a potential alternative is exemplified in fig5 . in this very rudimentary example we are determining the identities of 8 arrayed dinucleotides . the labeling of each dinucleotide is prior knowledge and consists of two labels , which are selected from a group of two distinct labels (“*” & amp ; “”). some of these labels are conjugated to a dinucleotide via a uv labile bond (“ o ”) which allows selective liberation of these labels ( 76 - 79 ). the dinucleotides are easily identified by simple comparison of the quantitative or qualitative signals before and after irradiation . in general the labeling scheme involves a multiply labeled entity , and a subsequent step wherein a subset of these labels is selectively liberated , disabled or enabled . the disabling or enabling occur by the making and / or breaking of chemical bonds , and an example thereof would be the bleaching of a fluorescent dye . the term “ labels ” as used here is quite broad in that it includes not only those substances which emit or can be induced to emit signals , but also includes substances which can appreciably alter the signals of an adjacent label . good examples of labels are fluorescent dyes , fluorescent energy transferers , fluorescent quenchers . these examples and accompanying figures have deliberately been made exceptionally simple so as to clearly and concisely present the invention . further information can be found in the accompanying u . s . provision patent application no . 60 / 296337 . many modifications and variations of the present invention are possible , and it is intended that all such modifications and variations be included within the scope of present invention as defined by the claims . the following articles are incorporated in their entirety by reference . they more fully describe the state of the art , and teach applicable material and methods . 1 ) a universal nucleoside for use at ambiguous sites in dna primers . nichols , et al nature 1994 june 9 ; 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