WO1999036567A2 - Procede permettant d'ameliorer la mise en oeuvre d'une distinction entre des appariements parfaits et des mesappariements au moyen d'une adn ligase modifiee - Google Patents

Procede permettant d'ameliorer la mise en oeuvre d'une distinction entre des appariements parfaits et des mesappariements au moyen d'une adn ligase modifiee Download PDF

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Publication number
WO1999036567A2
WO1999036567A2 PCT/US1999/000176 US9900176W WO9936567A2 WO 1999036567 A2 WO1999036567 A2 WO 1999036567A2 US 9900176 W US9900176 W US 9900176W WO 9936567 A2 WO9936567 A2 WO 9936567A2
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Prior art keywords
probes
sequence
probe
target
dna
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PCT/US1999/000176
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English (en)
Inventor
Narayan Baidya
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Hyseq, Inc.
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Priority to AU25577/99A priority Critical patent/AU2557799A/en
Publication of WO1999036567A2 publication Critical patent/WO1999036567A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention provides a method for detecting a target nucleic acid species including the steps of providing an array of probes affixed to a substrate and a plurality of labeled probes wherein each labeled probe is selected to have a first nucleic acid sequence which is complementary to a first portion of a target nucleic acid and wherein the nucleic acid sequence of at least one probe affixed to the substrate is complementary to a second portion of the nucleic acid sequence of the target, the second portion being adjacent to the first portion; applying a target nucleic acid to the array under suitable conditions for hybridization of probe sequences to complementary sequences; introducing a labeled probe to the array; hybridizing a probe affixed to the substrate to the target nucleic acid; hybridizing the labeled probe to the target nucleic acid; affixing the labeled probe to an adjacently hybridized probe in the array; and detecting the labeled probe affixed to the probe in the array.
  • a method for detecting a target nucleic acid of known sequence comprising the steps of: contacting a nucleic acid sample with a set of immobilized oligonucleotide probes attached to a solid substrate under hybridizing conditions wherein the immobilized probes are capable of specific hybridization with different portions of said target nucleic acid sequence; contacting the target nucleic add with a set of labelled oligonucleotide probes in solution under hybridizing conditions wherein the labeled probes are capable of spedfic hybridization with different portions of said target nudeic add sequence adjacent to the immobilized probes; covalently joining the immobilized probes to labelled probes that are immediately adjacent to the immobilized probe on the target sequence (e.g., with ligase); removing any non-ligated labelled probes detecting the presence of the target nuddc acid by detecting the presence of said labdled probe attached to the immobilized probes.
  • the present invention provides for an array of oligonucleotide probes comprising a nylon membrane; a plurality of subarrays of oligonucleotide probes on the nylon membrane, the subarrays comprising a plurality of individual spots wherein each spot is comprised of a plurality of oligonucleotide probes of the same sequence; and a plurality of hydrophobic barriers located between the subarrays on the nylon membrane, whereby the plurality of hyydrophobic barriers prevents cross contamination between adjacent subarrays.
  • the present invention provides a method for sequencing a repetitive sequence, having a first end and a second end, in a target nucleic add comprising the steps of: (a) providing a plurality of spacer oligonudeotides of varying lengths wherein the spacer oligonudeotides comprise the repetitive sequence; (b) providing a first oligonucleotide that is known to be adjacent to the first end of the repetitive sequence (c) providing a plurality of second oligonucleotides one of which is adjacent to the second end of the repetitive sequence, wherein the plurality of second oligonudeotides is labeled; (d) hybridizing the first and the plurality of second oligonucleotides, and one of the plurality of spacer oligonucleotides to the target nuddc add ; (e) ligating the hybridized oligonucleotides; (f) separating ligated oligonudeotides from unligated oligon
  • the present invention provides a method for sequencing a branch point sequence, having a first end and a second end, in a target nuddc add comprising the steps of: (a) providing a first oligonudeotide that is complementary to a first portion of the branch point sequence wherein the first oligonudeotide extends from the first end of the branch point sequence by at least one nudeotide; (b) providing a plurality of second oligonucleotides that are labeled, and are complementary to a second portion of the branch point sequence wherein the plurality of second oligonudeotides extend from the second end of the branch point sequence by at least one nucleotide, and wherein the portion of the second oligonudeotides that extend from the second end of the branch point sequence comprise sequences that are complementary to a plurality of sequences that arise from the branch point sequence (c) hybridizing the first oligonudeotide, and one of the plurality of second oligonucleo
  • the present invention provides a method for confirming a sequence by using probes that are predicted to be negative for the target nuddc add. The sequence of a target is then confirmed by hybridizing the target nucldc acid to the "negative" probes to confirm that these probes do not form perfect matches with the target nuddc add. Still further, the present invention provides a method for analyzing a nudeic add using oligonudeotide probes that are complexed with different labels so that the probes may be multiplexed in a hybridization reaction without a loss of sequence information (i.e., different probes have different labels so that hybridization of the different probes to the target can be distinguished).
  • the labels are radioisotopes, or floursecent molecules, or enzymes, or electrophore mass labds.
  • the differently labeled oligonucleotides probes are used in format III SBH, and multiple probes (more than two, with one ptrobe being the immobilized probe) are ligated together.
  • the present invention provides a method for detecting the presence of a target nuddc add having a known sequence when the target is present in very small amounts compared to homologous nuddc acids in a sample.
  • the target nuddc acid is an allele present at very low frequency in a sample that has nucldc acids from a large number of sources.
  • the target nucldc add has a mutated sequence, and is present at very low frequency within a sample of nuddc acids.
  • the present invention provides a method for confirming the sequence of a target nucleic acid by using single pass gd sequencing.
  • Primers for single pass gel sequencing are derived from the sequence obtained by SBH, and these primers are used in standard Sanger sequencing reactions to provide gel sequence information for the target nuddc add. The sequence obtained by single pass gel sequencing is then compared to the SBH derived sequence to confirm the sequence.
  • the present invention provides a method for solving branch points by using single pass gd sequencing.
  • Primers for the single pass gel sequencing reactions are identified from the ends of the Sfs obtained after a first round of SBH sequencing, and these primers are used in standard Sanger-sequencing reactions to provide gel sequencing information through the branch points of the Sfs.
  • Sfs are then aligned by comparing the Sanger-sequencing results through the branch points to the Sfs to identify adjoining Sfs.
  • the present invention provides for a method of preparing a sample containing target nucleic adds by PCR, without purifying the PCR products prior to the SBH reactions.
  • crude PCR products are applied to a substrate without prior purification, and the substrate may be washed prior to introduction of the labeled probes.
  • the present invention provides a method and an apparatus for analyzing a target nucldc acid.
  • the apparatus comprises two a ⁇ ays of nucldc adds that are mixed together at the desired time.
  • the nucldc acids in one of the a ⁇ ays are labded.
  • a material is disposed between the two arrays and this material prevents the mixing of nucldc adds in the arrays. When this material is removed, or rendered permeable, the nuddc adds in the two a ⁇ ays are mixed together.
  • the nucldc adds in one array are target nucldc adds and the nucleic acids in the other are oligonu eotide probes. In another preferred embodiment, the nuddc acids in both arrays are oligonucleotide probes.
  • the nucldc acids in one array are oligonudeotide probes and target nucldc acids, and nuddc adds in the other array axe oligonudeotide probes.
  • the nuddc adds in both arrays are oligonucleotide probes and target nucleic adds.
  • One method of the present invention using the apparatus described above comprises the steps of providing an array of nucldc acids fixed to a substrate, providing a second array of nucleic adds, providing conditions that allow the nuddc acids in the second array to come into conuct with the nuddc acids of the fixed array wherein one of the arrays of nuddc acids are target nucldc acids and the other array is oligonucleotide probes, and analyzing the hybridization results.
  • the fixed array is target nucleic acid and the second array is labeled oligonucleotide probes.
  • a second method of the present invention using the apparatus described above comprises the steps of providing two arrays of nuddc add probes, providing conditions that allow the two a ⁇ ays of probes to come into contact with each other and a target nuddc acid, ligating together probes that are adjacent on the target nucldc acid, and analyzing the results.
  • the probes in one array are fixed and the probes in the other array are labded.
  • the present invention provides substrates on which arrays of oligonudeotide probes are fixed, wherein each probe is separated from its neighboring probes by a physical barrier that is resistant to the flow of the sample solution.
  • the physical barrier is made of a hydrophobic material.
  • the present invention provides a method for making the arrays of oligonudeotide probes that are separated by physical barriers.
  • a grid is applied to the substrate using an ink-jet head that applies a material which reduces the reaction volume of the array.
  • the present invention provides substrates on which oligonucleotides are fixed to form a three-dimensional array.
  • the three-dimensional array combines high resolution for reading probe results (each levd has a relativdy low density of probes per cm 2 ), with high information content in three dimensional space (multiple levels or probes).
  • the present invention provides a substrate to which oligonudeotide probes are fixed, wherein the oligonudeotide probes have spacers, and wherein the spacers increase the distance between the substrate and the informational portion of the oligonucleotide probe (e.g., the portion of the oligonudeotide probe which binds to the target and gives sequence information).
  • the spacer comprises ribose sugars and phosphates, wherein the phosphates covalently bind the ribose sugars into a polymer by forming esters with the ribose sugars through thdr 5' and 3' hydroxyl groups.
  • the physical property includes any that can be used to differentiate the discrete partides, and includes, for example, size, flourescence, radioactivity, electromagnetic charge, or absorbance, or label(s) may be attached to the particle such as a dye, a radionuclide, or an EML.
  • discrete particles are separated by a flow cytometer which detects the size, charge, flourescence, or absorbance of the mixturee.
  • the change in physical condition or addition of an agent enhances discrimination in a number of ways, for example, the physical condition or agent may increase the difference in the on rates or off rates between a perfect match product and a mismatch product (a kinetic effect); or the reaction time may be decreased so that binding of the probe to a perfect match site and/or a mismatch site does not reach equilibrium; or the physical condition or agent may increase the binding energy difference between a perfect match and a mismatch (a free energy [ ⁇ G] effect); or the physical condition or agent may enhance the discrimination effect of another agent or physical condition ( ⁇ G or kinetic effect); or the physical condition or agent may preferentially modify the perfect match or mismatch complexes formed between complementary polynucleotides; or the physical condition or agent may enhance the discrimination of the physical condition or agent which physically modifies the complexed polynudeotides ( ⁇ G, kinetic, or conformational effect); or some combination of these and other factors.
  • the physical condition or agent may increase the difference in the on rates or off rates between a
  • the agent, agents or physical condition(s) modify the activity of a protein which binds to and/or modifies the complexed or uncomplexed nudeic adds.
  • the agent is one of those recited supra.
  • the physical condition is sdected from the group comprising temperature, pH, ionic strength, time, and/or others such as, e.g., those listed in The Handbook of Chemistry and Physics, CRC Press.
  • Serial scoring of thousands of samples on large a ⁇ ays may be performed in thousands of independent hybridization reactions using small pieces of membranes.
  • the identification of DNA may involve 1-20 probes per reaction and the identification of mutations may in some cases involve more than 1000 probes specifically selected or designed for each sample.
  • specific probes may be synthesized or sdected for each mutation detected in the first round of hybridizations.
  • DNA samples may be prepared in small a ⁇ ays which may be separated by appropriate spacers, and which may be simultaneously tested with probes selected from a set of oligonucleotides which may be arrayed in multiwell plates. Small arrays may consist of one or more samples.
  • DNA samples in each small array may include mutants or individual samples of a sequence.
  • Consecutive small arrays may be organized into larger arrays, Such larger arrays may include replication of the same small array or may indude arrays of samples of different DNA fragments.
  • a universal set of probes indudes sufficient probes to analyze a DNA fiagment with prespecified predsion, e.g. with respect to the redundancy of reading each base pair ("bp"). These sets may include more probes than are necessary for one spedfic fragment, but may indude fewer probes than are necessary for testing thousands of DNA samples of different sequence.
  • DNA or allele identification and a diagnostic sequencing process may indude the steps of: 1) Sdection of a subset of probes from a dedicated, representative or universal set to be hybridized with each of a plurality of small arrays;
  • a small set of shorter probes may be used in place of a longer unique probe.
  • a universal set of probes may be synthesized to cover any type of sequence. For example, a full set of 6-mers indudes only 4,096 probes, and a complete set of 7- ⁇ ners includes only 16,384 probes.
  • Full sequencing of a DNA fragment may be performed with two levds of hybridization.
  • One levd is hybridization of a sufficient set of probes that cover every base at least once.
  • a spedfic set of probes may be synthesized for a standard sample. The results of hybridization with such a set of probes reveal whether and where mutations (differences) occur in non-standard samples. Further, this set of probes may indude "negative" probes to confirm the hybridization results of the "positive" probes. To determine the identity of the changes, additional spedfic probes may be hybridized to the sample.
  • an a ⁇ ay of sample avoids consecutive scoring of many digonudeotides on a single sample or on a small set of samples. This approach allows the scoring of more probes in parallel by manipulation of only one physical object.
  • Subarrays of DNA samples 1000 bp in length may be sequenced in a relativdy short period of time. If the samples are spotted at 50 subarrays in an array and the a ⁇ ay is reprobed 10 times, 500 probes may be scored. In screening for the occurrence of a mutation, enough probes may be used to cover each base three times. If a mutation is present, several covering probes will be affected. The use of information about the identity of negative probes may map the mutation with a two base precision.
  • an additional 15 probes may be employed. These probes cover any base combination for two questionable positions (assuming that ddetions and insertions are not involved). These probes may be scored in one cyde on 50 suba ⁇ ays which contain a given sample. In the implementation of a multiple label color scheme (i.e., multiplexing), two to six probes, each having a different label such as a different fluorescent dye, may be used as a pool, thereby reducing the number of hybridization cycles and shortening the sequencing process. In more complicated cases, there may be two dose mutations or insertions. They may be handled with more probes. For example, a three base insertion may be solved with 64 probes. The most complicated cases may be approached by several steps of hybridization, and the selecting of a new set of probes on the basis of results of previous hybridizations.
  • subarrays to be analyzed include tens or hundreds of samples of one type, then several of them may be found to contain one or more changes (mutations, insertions, or ddetions). For each segment where mutation occurs, a specific set of probes may be scored. The total number of probes to be scored for a type of sample may be several hundreds. The scoring of replica a ⁇ ays in parallel facilitates scoring of hundreds of probes in a relatively small number of cydes. In addition, compatible probes may be pooled. Positive hybridizations may be assigned to the probes selected to check particular DNA segments because these segments usually differ in 75% of their constituent bases.
  • targets may be analyzed. These targets may represent pools of fragments such as pools of exon dones.
  • a spedfic hybridization scoring method may be employed to define the presence of mutants in a genomic segment to be sequenced from a diploid chromosomal set. Two variations are where: i) the sequence from one chromosome represents a known allde and the sequence from the other represents a new mutant; or, ii) both chromosomes contain new, but different mutants. In both cases, the scanning step designed to map changes gives a maximal signal difference of two-fold at the mutant position. Further, the method can be used to identify which alleles of a gene are carried by an individual and vvhether ih& individual is homozygous or heterozygous for that gene.
  • Scoring two-fold signal differences required in the first case may be achieved efficiently by comparing corresponding signals with homozygous and heterozygous controls.
  • This approach allows determination of a relative reduction in the hybridization signal for each particular probe in a given sample- This is significant because hybridization efficiency may vary more than two-fold for a particular probe hyb ridized with different nucldc add fragments having the same full match target.
  • di: ⁇ erent mutant sites may affect more than one probe depending upon the number of oligonuclei itide probes. Decrease of the signal for two to four consecutive probes produces a more signi Scant indication of a mutant site. Results may be checked by testing with small sets of selected ] .robes among which one or few probes selected to give a full match signal which is on average d ght-fold stronger than the signals coming from mismatch-containing duplexes.
  • Partitioned me nbranes allow a very flexible organization of experiments to accommodate rdatively larger numb as of samples representing a given sequence type, or many different types of samples represented w ith relatively small numbers of samples.
  • a range of 4-256 samples can be handled with particula ⁇ efficiency.
  • Subarrays within this range of numbers of dots may be designed to match the configura a ion and size of standard multiwell plates used for storing and labeling oligonu eotides.
  • the : size of the subarrays may be adjusted for different number of samples, or a few standard suba ⁇ ay ⁇ : ;izes may be used.
  • a nui Jdc acid to be sequenced or intermediate fragments thereof may be applied to the first set of probes in double-stranded form (especially where a recA protein is present to permit hybridization un ier non-denaturing conditions), or in single-stranded form and under conditions which permi : hybrids of different degrees of complementarity (for example, under conditions which allow discrimination between full match and one base pair mismatch hybrids).
  • the nuddc acid to be s quenced or intermediate fragments thereof may be applied to the first set of probes before, after or simultaneously with the second set of probes.
  • nudeotide bases "match” or are "complementary” if they form a stable duplex by hydrogen bonding under specified conditions.
  • adenine matches thymine (“T), but not guanine (“G”) or cytosine ("C”).
  • G matches C, but not A or T.
  • Other bases which will hydrogen bond in less spedfic fashion such as inosine or the Universal Base (“M” base, Nichols et al 1994), or other modified bases, such as methylated bases, for example, are complementary to those bases for which they foim a stable duplex under spedfied conditions.
  • a probe is said to be “perfedly complementary” or is said to be a "perfect match” if each base in the probe forms a duplex by hydrogen bonding to a base in the nuddc acid to be sequenced according to the Watson and Crick base paring rules (i.e., absent any su ⁇ ounding sequence effects, the duplex formed has the maximal binding energy for a particular probe).
  • Perfectly complementary and “perfect match” are also meant to encompass probes which have analogs or modified nucleotides.
  • a list of probes may be assembled wherein each probe is a perfect match to the nudeic acid to be sequenced.
  • the probes on this list may then be analyzed to order them in maximal overlap fashion. Such ordering may be accomplished by comparing a first probe to each of the other probes on the list to determine which probe has a 3' end which has the longest sequence of bases identical to the sequence of bases at the 5' end of a second probe.
  • the first and second probes may then be overlapped, and the process may be repeated by comparing the 5' end of the second probe to the 3' end of all of the remaining probes and by comparing the 3' end of the first probe with the 5' end of all of the remaining probes.
  • the process may be continued until there are no probes on the list which have not been overlapped with other probes. Altemativdy, more than one probe may be sdected from the list of positive probes, and more than one set of overlapped probes ("sequence nucleus") may be generated in paralld. ' The list of probes for either such process of sequence assembly may be the list of all probes which are perfectly complementary to the nucleic add to be sequenced or may be any subset thereof.
  • Hybridization and washing conditions may be selected to detect substantially perfect match hybrids (such as those wherein the fiagment and probe hybridize at six out of seven positions), may be selected to allow differentiation of perfect matches and one base pair mismatches, or may be selected to permit detection only of perfect match hybrids.
  • ligation may be implemented by a chemical ligating agent (e.g. water-soluble caibodii ide or cyanogen bromide), or a ligase enzyme, such as the commercially available T 4 DNA ligase may be employed.
  • a chemical ligating agent e.g. water-soluble caibodii ide or cyanogen bromide
  • a ligase enzyme such as the commercially available T 4 DNA ligase may be employed.
  • the washing conditions may be selected to distinguish between adjacent versus nonadjacent labded and immobilized probes exploiting the difference in stability for adjacent probes versus nonadjacent probes.
  • oligonucleotides are attached to a glass surface using a modified protocol from Zehn Gao et d., Nucl. Acids. Res. (1994) 22:5456-5465.
  • the glass surface is activated by adding an amino-silane functional group, that is coupled with a phenyldiisothiocyanate (DITC).
  • DITC phenyldiisothiocyanate
  • 5 '-amino oligonucleotides are attached to this glass substrate by spotting onto the DITC activated glass surface and incubating for one hour al 37 °C in a humid chamber.
  • the substrate which supports the array of oligonucleotide probes is partitioned into sections so that each probe in the array is separated from adjacent probes by a physical barrier which may be, for example, a hydrophobic material.
  • the physicd barrier has a width of from 100 ⁇ m to 30 ⁇ m. In a more preferred embodiment, the distance from the center of each probe to the center of any adjacent probes is 325 ⁇ m.
  • a reusable Format 3 SBH array may be produced by introducing a deavable bond between the fixed and labeled probes and then cleaving this bond after a round of Format 3 analyzes is finished.
  • the labeled probes may be ribonucleotides or a ribonudeotide may be used as the joining base in the labded probe so that this probe may subsequently be removed, e.g., by RNAse or ura l-DNA glycosylate treatment, or NaOH treatment
  • bonds produced by chemicd ligation may be sdectivdy cleaved.
  • cyding hybridizations to increase the hybridization signal, for example by performing a hybridization cyde under conditions (e.g. temperature) optimally selected for a first set of labeled probes followed by hybridization under conditions optimally sdected for a second set of labeled probes. Shifts in reading frame may be determined by using mixtures (preferably mixtures of equimolar amounts) of probes ending in each of the four nucleotide bases A, T, C and G.
  • SBH is a well developed technology that may be practiced by a number of methods known to those skilled in the art. Spedfically, techniques related to sequendng by hybridization of the following documents is incorporated by reference herein: Drmanac et al., U.S. Patent No. 5,202,231 (hereby incorporated by reference herein) - Issued April 13, 1993; Drmanac et al., Genomics, 4, 114-128 (1989); Drmanac et al., Proceedings of the First Int'l Conf. Electrophoresis Superco puting Human Genome Cantor et al. eds, World Scientific Pub.
  • EMF expression modulating fragment
  • Sets of probes may also be comprised of from 50 probes to a universal set of probes (all probes of a certain length), more preferably the set is comprised of 100-500 probes, and in a most preferred embodiment, the probe set contains 300 probes.
  • the set of probes are 6-9 nucleotides in length, and are used to duster cDNA clones into groups of similar or identical sequences, so that single representative clones may be selected from each group for sequencing.
  • Probes may be prepared using standard chemistry with one to three non-specified (mixed A,T,C and G) or universal (e.g. M base or inosine) bases at the ends. If radidabelling is used, probes may have an OH group at the 5' end for kinasing by radiolabdled phosphorous groups.
  • probes labdled with any compatible system such as fluorescent dyes
  • any compatible system such as fluorescent dyes
  • Other types of probes such as PNA (Protdn Nucleic Adds)or probes containing modified bases which change duplex stability dso may be used.
  • Probes may be stored in bar-coded multiwdl plates. For small numbers of probes, 96-well plates may be used; for 10,000 or more probes, storage in 384- or 864-wdl plates is preferred.
  • Stacks of 5 to 50 plates are enough to store all probes. Approximatdy 5 pg of a probe may be suffident for hybridization with one DNA sample. Thus, from a small synthesis of about 50 mg per probe, ten million samples may be analyzed. If each probe is used for every third sample, and if each sample is 1000 bp in length, then over 30 billion bases (10 human genomes) may be sequenced by a set of 5,000 probes.
  • Modified oligonudeotides may be introduced into hybridization probes and used under appropriate conditions therefor.
  • pyrimidines with a hdogen at the exposition may be used to improve duplex stability by influencing base stacking.
  • 2,6-diaminopurine may be used to provide a third hydrogen bond in base pairing with thymine, thereby thermdly stabilizing DNA-duplexes.
  • 2,5-diaminopurine may increase duplex stability to allow more stringent conditions for annealing, thereby improving the spedfidty of duplex formation, suppressing background problems and permitting the use of shorter oligomers.
  • This new analogue, l-(2 -deoxy- -D-ribfuranosyl)-3-nitropy ⁇ ole (designated M) was generated for use in oligonudeotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available.
  • This analogue maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex.
  • the M nudeoside analogue was designed to maximize stacking interactions using aprotic polar substituents linked to het ⁇ roaromatic rings, enhancing intra- and inter-strand stacking interactions to lessen the role of hydrogen bonding in base-pairing spedfidty.
  • Nichols et al. (3994) favored 3-nitropy ⁇ ole 2 -deoxyribonucleoside because of its structura] and dectronic resemblance to p-nitroaniline, whose derivatives are among the smallest known intercalators of double-stranded DNA.
  • the dimethoxytrityl-protected phosphoramidite of nudeoside M is dso available for incorporation into nudeotides used as primers for sequencing and polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • M has a unique property of its ability to replace long strings of contiguous nucleosides and still yield fiinctiond sequencing primers. Sequences with three, six and nine M substitutions have all been reported to give readable sequencing ladders, and PCR with three different M-containing primers all resulted in amplification of the correct product (Nichols et al, 1994).
  • the sets of probes to be hybridized in each of the hybridization cycles on each of the subarrays is defined. For example, a set of 384 probes may be sdected from the universal set, and 96 probings may be performed in each of 4 cycles. Probes sdected to be hybridized in one cycle preferably have similar Cr+C contents.
  • Selected probes for each cycle are transferred to a 96-well plate and then are labdled by kinasing or by other labeling procedures if they are not labdled (e.g. with stable fluorescent dyes) before they are stored.
  • a new set of probes may be defined for each of the subarrays for additional cycles. Some of the a ⁇ ays may not be used in some of-the cycles. For example, if only 8 of 64 patient samples exhibit a mutation and 8 probes are scored first for each mutation, then dl 64 probes may be scored in one cycle and 32 subarrays are not used. These unused subarrays may then be treated with hybridization buffer to prevent drying of the filters.
  • Probes may be retrieved from the storing plates by any convenient approach, such as a single channd pipetting device, or a robotic station, such as a Beckman Biom ⁇ k 1000 (Beckman Instruments, FuUerton, California) or a Mega Two robot (Megamation, Lawre ⁇ ceville, New Jersey), A robotic station may be integrated with data andysis programs and probe managing programs. Outputs of these programs may be inputs for one or more robotic stations. Probes may be retrieved one by one and added to subarrays covered by hybridization buffer.
  • retrieved probes be placed in a new plate and labelled or mixed with hybridization buffer.
  • the preferred method of retrieval is by accessing stored plates one by one and pipetting (or transferring by metal pins) a sufficient amount of each selected probe from each plate to spedfic wdls in an intermediary plate. Axi a ⁇ ay of individudly addressable pipe ⁇ es or pins may be used to speed up the retrievd process.
  • the oligonudeotide probes may be prepared by automated synthesis, which is routine to those of skill in the art, for example, using and Applied Biosystems system. Altemativdy, probes may be prepared using Genosys Biotechnologies Inc. Methods using stacks of porous Teflon wafers.
  • Oligonucleotide probes may be labded with, for example, radioactive labds ( ⁇ S, 32 P, 33 P, and preferably, 33 P) for arrays with 100-200 um or 100-400 um spots; non-radioactive isotopes (Jacobsen et al, 1990); or fluorophores (Brumbaugh et al, 1988). All such labding methods are routine in the art, as exemplified by the relevant sections in Sambrook et al ( 1989) and by further references such as Schubert etal. (1990), Murakami et al. (1991) and Cate e/ ⁇ /. (1991), dl artides being spedficdly incorporated herein by reference.
  • radiolabelling the common methods are end-labeling using T4 polynucleotide kinase or high specific activity labeling using Klenow or even T7 polymerase. These are described as follows.
  • Synthetic oligonudeotides are synthesized without a phosphate group at their 5 termini and are therefore easily labded by transfer of the - 32 P or - 3 P from [ - 32 P]ATP or [ - 33 P]ATP using the enzyme bacteriophage T4 polynucleotide kinase. If the reaction is carried out effidently, the spedfidty activity of such probes can be as high as the spedfic activity of the [ - P]ATP or [ - 33 P] ATP itsdf .
  • the reaction described bdow is designed to label 10 pmoles of an oligonucleotide to high specific activity. Labding of different amounts of oligonudeotide can easily be achieved by increasing or decreasing the size of the reaction, keeping the concentrations of all components constant.
  • fluorescent labding of an oligonucleotide at its 5'-end initially involved two steps. First, a N-protected aminodkyl phosphoramidite derivative is added to the 5'-end of an oligonudeotide during automated nucldc add synthesis. After r ⁇ movd of dl protecting groups, the NHS ester of an appropriate fluorescent dye is coupled to the 5'-amino group overnight followed by purification of the labeled oligonudeotide from the excess of dye using reverse phase HPLC or PAGE. Schubert et al. (1990) described the synthesis of a phosphoramidite that enables oligonudeotides labded with fluorescein to be produced during automated DNA synthesis.
  • Cate et al (1991) describe the use of oligonudeotide probes directly conjugated to alkdine phosphatase in combination with a direct chemiluminescent substrate (AMPPD) to dlow probe detection.
  • AMPPD direct chemiluminescent substrate
  • labds include ligands which can serve as specific binding members to a labeled antibody, chemiluminescers, enzymes, antibodies which can serve as a specific binding pdr member for a labeled ligand, and the like.
  • ligands which can serve as specific binding members to a labeled antibody
  • chemiluminescers enzymes
  • antibodies which can serve as a specific binding pdr member for a labeled ligand, and the like.
  • a wide variety of labds have been employed in immunoassays which can readily be employed, Still other labels include antigens, groups with specific reactivity, and ele ⁇ rochemicdly detectable modties.
  • EMLs are detected using a variety of well known electron capture mass spectrometry devices (e.g., devices sold by Finnigan Corporation). Further, techniques that may be used in the detection of EMLs indude, for example, fast atomic bombardment mass spectrometry (see, e.g., Koster et al, Biomedicd Environ. Mass Spec. 14:111- 116 (1987)); plasma desorption mass spectrometry; dectrospray/ionspray (see, e.g., Fenn et al., J. Phys. Chem. 88:4451-59 (1984), PCT Appln. No. WO 90/14148, Smith et ⁇ rf. And. Chem.
  • fast atomic bombardment mass spectrometry see, e.g., Koster et al, Biomedicd Environ. Mass Spec. 14:111- 116 (1987)
  • plasma desorption mass spectrometry see, e.g., F
  • the oligonudeotide terminus must have a S'-end phosphate group. It is, perhaps, even possible for biotin to be covdently bound to CovaLink and then streptavidin used to bind the probes.
  • the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-Mdm 7 ), is then added to a find concentration of 10 mM 1-Melm 7 . A ss DNA solution is then dispensed into CovaLink NH strips (75 ul well) standing on ice. Carbodiimide 0.2 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (BDC), dissolved in
  • aminimd concentration of the probes may be used and hybridization time extended to the maximd practical levd.
  • knowledge of a "normd" sequence dlows the use of the continuous stacking interaction phenomenon to increase the signd.
  • additiond unlabelled probes which hybridize back to back with a labdled one may be added in the hybridization reaction. The amount of the hybrid may be increased several times.
  • the probes may be connected by ligation. This approach may be important for resdving DNA regions forming "compressions".
  • images of the filters may be obtained, preferably by phosphorstorag ⁇ technology.
  • Fluorescent labds may be scored by CCD cameras, confoca] microscopy or otherwise.
  • raw signds are normalized based on the amount of target in each dot Differences in the amount of target DNA per dot may be corrected for by dividing signds of each probe by an average signd for dl probes scored on one dot.
  • the normdized signds may be scded, usually from 1-100, to compare data from different experiments.
  • control DNAs may be used to determine an average background signd in those samples which do not contain a full match target.
  • homozygptic controls may be used to allow recogmtion of heterozygotes in the samples.
  • Applied Biosystems 381 A DNA synthesizex. Most of the probes used were not purified by HPLC or gel electrophoresis. For example, probes were designed to have both a single perfectly complementary target in interferon, a Ml 3 clone containing a 921 bp Eco RI-Bgl II human B 1 - interferon fragment (Ohno and Tangiuchi, Proc. Natl. Acad. Sd. 74: 4370-4374 (1981)], and at least one target with an end base mismatch in Ml 3 vector itself.
  • oligonucleotides End labeling of oligonucleotides was performed as described [Maniatis et d., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, New York (1982)] in 10 ml containing T4-polynudeotide kinase (5 units Amersham), ⁇ -ATP (3.3 pM, 10 mCi Amersham 3000 Ci/mM) and oligonucleotide (4 pM, 10 ng). Spedfic activities of the probes were 2.5-5 X 109 cpmraM.
  • the dots were exdsed from the dried filters after autoradiography [a phosphoimager (Molecdar Dynamics, Sunnyvde, California) may be used] placed in liquid scintillation cocktdl and counted.
  • the uncorrected ratio of cpms for IF and M13 dots is given as D.
  • the equilibrium may be moved towards hybrid formation by increasing probe concentration and/or decreasing temperature, however, during washing cydes in large volumes of buffer, the melting reaction is dominant and the back reaction hybridization is insignificant, since the probe is absent. This andysis indicates workable Short Oligonucleotide Hybridization (SOH) conditions call be varied for probe concentration or temperature.
  • SOH Short Oligonucleotide Hybridization
  • the background, B represents the lowest hybridization signd detectable in the system. Since any further decrease of H, may not be examined, D increases upon continued washing.
  • the hybridization signd depends on the amount of target available on the filter for reaction with the probe. A necessary control is to show that the difference in sign intensity is not a reflection of varying amounts of nuddc acid in the two dots.
  • Hybridization with a probe that has the same number and kind of targets in both IF and M13 shows that there is an equal amount of DNA in the dots. Since the efficiency of hybrid formation increases with hybrid length, the signal for a duplex having six nudeotides was best detected with a high mass of oligonucleotide target bound to the filter.
  • oligonucleotide target molecdes Due to thrir lower molecular wdght, a larger number of oligonucleotide target molecdes can be bound to a given surface area when compared to large molecdes of nucldc aci that serves as target.
  • the M13 system has the advantage of showing the effects of target DNA complexi ⁇ y on the levds of discrimination. For two octamers having either none or five mismatched targets and differing in ody one GC pair the observed discriminations were 18.3 and 1.7, respectivdy.
  • three probes 8 nucleotides in length were tested on a collection of 1 plasmid DNA dots made from a library in Bluescript vector. One probe was present and specific for Bluescript vector but was absent in M13, while the other two probes had targets that were inserts of known sequence. This system dlowed the use of hybridization negative or positive control DNAs with each probe. This probe sequence (CTCCCTTT) dso had a complementary target in the interferon insert.
  • nudeic acid for this purpose are present in convenient biologicd samples such as a few microiiters of Ml 3 culture, a plasmid prep from 10 ml of ba erid culture or a single colony of bacteria, or less than 1 ml of a standard PCR reaction.
  • the hybridization conditions described in this invention for short oligonudeotide hybridization using low temperatures give better discriminating for dl sequences and duplex hybrid inputs.
  • the only price pad in achieving uniformity in hybridization conditions for different sequences is an increase in washing time from minutes to up to 24 hours depending on the sequence. Moreover, the washing time can be further reduced by decreasing the sdt concentration.
  • An a ⁇ ay of subarrays dlows for effident sequencing of a small set of samples arrayed in the form of replicated subarrays For example, 64 samples may be axrayed on a 8 X 8 mm subarray and 16 X 24 subarrays may be replicated on a 15 X 23 cm membrane with 1 mm wide spacers between the subarrays.
  • Several replica membranes may be made, For example, probes from a universd set of three thousand seventy-two 7-mers may be divided in thiity-two 96-wdl plates and labelled by kinasing. Four membranes may be processed in paralld during one hybridization cycle. On each membrane, 384 probes may be scored.
  • All probes may be scored in two hybridization cycles. Hybridization intensities may be scored and the sequence assembled as described below. If a single sample subarray or subarrays contains several unknowns, especidly when similar samples are used, a smdler number of probes may be suffident if they are intelligently sdected on the basis of resdts of previously scored probes. For example, if probe AAAAAAA is not positive, there is a smdl chance that any of 8 overlapping probes are positive, If AAAAAAA is positive, then two probes are usudly positive.
  • the sequencing process in this case consists of first hybridizing a subset of ⁇ iinimdly overlapped probes to define positive anchors and then to successivdy select probes which confirms one of the most likely hypotheses about the order of anchors and size and type of gaps between them.
  • pools of 2-10 probes may be used where each probe is sdected to be positive in ody one DNA sample which is different from the samples expected to be positive with other probes from the pool.
  • the subarray approach dlows efficient implementation of probe competition (overlapped probes) or probe cooperation (continuous stacking of probes) in sdving branching problems.
  • the sequence assembly program determines candidate sequence subfragments (SFs).
  • SFs candidate sequence subfragments
  • additiond information has to be provided (from overlapped sequences of DNA fragments, similar sequences, single pass gel sequences, or from other hybridization or restriction mapping data).
  • Primers for single pass gel sequencing through the branch points are identified from the SBH sequence info ⁇ nation or from known vector sequences, e.g., the flanking sequences to the vector insert site, and standard Sanger- sequencing reactions are performed on the sample DNA. The sequence obtained from this sing!
  • e pass gel sequendng is compared to the Sfs that read into and out of the branch points to identify the order of the Sfs. Further, singe pass gel sequencing may be combined with SBH to de novo sequence or re-sequence a nudeic add.
  • each of 64 samples described in this example there are about 100 branching points, and if 8 samples axe andyzed in parallel in each subarray, then at least 800 subarray probings solve dl branches, This means that for the 3072 basic probings an additional 800 probings (25%) are employed. More preferably, two probings are used for one branching point. If the subanays are smdler, less additional probings are used. For example, if subarrays consist of 16 samples, 200 additional probings may be scored (6%). By using 7-mer probes (N ⁇ ⁇ ) and competitive or collaborative branching solving approaches or both, fragments of about 1000 bp fragments may be assembled by about 4000 probings.
  • NB 8 N 8-mer probes 4 kb or longer fragments may be assembled with 12,000 probings. Gapped probes, for example, NB 4 NB 3 N or NB 4 NB 4 N may be used to reduce the number of branching points.
  • Oligonudeotide probes having an informative length of four to 40 bases are synthesized by standard chemistry and stored in tubes or in multiwdl plates. Spedfic sets of probes comprising one to 10,000 probes are arrayed by deposition or in situ synthesis on separate supports or distinct sections of a larger support. In the last case, sections or subarrays may be separated by physical or hydrophobic barriers. The probe arrays may be prepared by in situ synthesis. A sample DNA of appropriate size is hybridized with one or more specific a ⁇ ays. Many samples may be inte ⁇ ogated as pools at the same subarrays or independently with different subarrays within one support.
  • a single labelled probe or a pool of labdled probes is added on each of the subarrays. If attached and labdled probes hybridize back to back on the complementary target in the sample DNA they are ligated. Occurrence of ligation will be measured by detecting a label from the probe.
  • This procedure is a variant of the described DNA andysis process in which DNA samples are not permanently attached to the support. Transient attachment is provided by probes fixed to the support. In this case there is no need for a target DNA arraying process. In addition, ligation dlows detection of longer oligonucleotide sequences by combining short labelled probes with short fixed probes.
  • the process has several unique features. Basicdly, the transient attachment of the target allows its reuse. After ligation occur the target may be released and the label will stay covdently attached to the support. This feature dlows cycling the target and production of detectable signd with a smdl quantity of the target. Under optimd conditions, targets do not need to be amplified, e.g. natural sources of the DNA samples may be directly used for diagnostics and sequencing purposes. Targets may be released by cyding the temperature between eff ⁇ rie ⁇ t hybridization and effident mdting of duplexes. More preferably, there is no cyding.
  • the temperature and concentrations of components may be defined to have an equilibrium between free targets and targets entered in hybrids at about 50:50% levd. In this case there is a continuous production of ligated products. For different purposes different eqdlibrium ratios are optimd.
  • An electric fidd may be used to enhance target use.
  • a horizontal field pdsing within each subarray may be employed to provide for faster target sorting.
  • the equilibrium is moved toward hybrid formation, and unlabdled probes may be used.
  • an appropriate washing (which may be helped by a vertical electric field for restricting movement of the samples) may be performed.
  • Several cycles of discriminative hybrid melting, target harvesting by hybridization and ligation and removing of unused targets may be introduced to increase specifidty.
  • labdled probes are added and v ⁇ rticd electricd pulses may be applied. By increasing temperature, an optimal free and hybridized target ratio may be achieved.
  • the vertical dectric fidd prevents diffusion of the sorted targets.
  • the subanays of fixed probes and sets of labelled probes may be arranged in various ways to allow an efficient and flexible sequencing and diagnostics process. For example, if a short fiagment (about 100-500 bp) of a bacterid genome is to be partially or completdy sequenced, smdl arrays of probes (5-30 bases in length) designed on the bases of known sequence may be used. If interrogated with a different pool of 10 labdled probes per subarray, an a ⁇ ay of 10 subarrays each having 10 probes, dlows checking of 200 bases, assuming that ody two bases connected by ligation are scored.
  • probes may be displaced by more than one base to cover the longer target with the same number of probes.
  • the target may be interrogated directly without amplification or isolation from the rest of DNA in the sample.
  • several targets may be andyzed (screened for) in one sample simdtaneously. If the obtained results indicate occurrence of a mutation (or a pathogen), additiond pools of probes may be used to detect type of the mutation or subtype of pathogen. This is a desirable feature of the process which may be very cost effective in preventive diagnosis where only a smdl fraction of patients is expected to have an infection or mutation.
  • various detection methods may be used, for example, ⁇ adiolabds, fluorescent labels, enzymes or antibodies (chemilu inescence), large olecdes or particles detectable by light scattering or interferometric procedures.
  • mismatch oligonudeotides In addition to the perfectly matching oligonucleotides, mismatch oligonudeotides, mismatdi oligonudeotides wherein intemd or end mismatches occur in the duplex formed by the oligonucleotide and the target were examined. In these andyses, the lowest practicd temperature was used to maximize hybridization formation. Washes were accomplished at the same or lower temperatures to ensure maximd discrimination by utilizing the greater dissociation rate of mismatch versus matched oligonucleotide/target hybridization. These conditions are shown to be applicable to dl sequences dthough the absolute hybridization yield is shown to be sequence dependent.
  • the least destabilizing mismatch that can be postdated is a simple end mismatch, so that the test of sequencing by hybridization is the ability to discriminate perfectly matched oligonudeotid ⁇ /target duplexes from end-mismatched oligonucleotide/target duplexes.
  • the discriminative vdues for 102 of 105 hybridizing oligonucleotides in a dot blot format were greater than 2 dlowing a highly accurate generation of the sequence. This system dso dlowed an analysis of the effect of sequence on hybridization formation and hybridization instability.
  • the set of 93 probes provided consecutive overlapping frames of the target sequence e displaced by one o ⁇ two bases.
  • hybridization was examined for 12 additiond probes that contained at least one end mismatch when hybridized to the 100 bp test target sequence. Also tested was the hybridization of twelve probes with target end-mismatched to four other control nucldc acid sequences chosen so that the 12 oligonucleotides formed perfectly matched duplex hybrids with the four control DNAs.
  • H p defines the amount of hybrid duplex formed between a test target and an oligonucleotide probe.
  • D Discrimination vdues were obtained where D was defined as the ratio of signd intensities between 1 ) the dot containing a perfect matched duplex fo ⁇ ned between test oligonudeotide and target or control nucleic acid and 2) the dot containing a mismatch duplex formed between the same oligonucleotide and a different site within the target or control nucldc add.
  • Variations in the vdue of D resdt from either 1) perturbations in the hybridization efficiency • which dlows visudization of signd over background, or 2) the type of mismatch found between the test oligonudeotide and the target.
  • the D vdues obtained in this experiment were between 2 and 40 for 102 of the 105 oligonudeotide probes examined. Cdcdations of D for the group of 102 digonucleotides as a whole showed the average D was 10.6.
  • E ⁇ or in the target for probes with low H p was exduded as a possibility because such an e ⁇ or would have affected the hybridization of each of the other right overlapping oligonudeotides. There was no apparent instability due to sequence mismatch for the other overlapping digonudeotides, indicating the target sequence was co ⁇ ect.
  • E ⁇ or in the oligonucleotide sequence was exduded as a possibility after the hybridization of seven newly synthesized oligonudeotides as re-examined. Only 1 of the seven oligonucleotides resdted in a better D vdue. Low hybrid formation vdues may result from hybrid instability or from an inabUity to form hybrid duplex.
  • the resdts indicate that reliable results may be obtained to generate sequences if octamer and nonamer oligonucleotides are used. These results show that using the methods described long sequences of any specific target nucleic acid may be generated by maximd and unique overlap of constituent oligonucleotides. Such sequencing methods are dependent on the content of the individud component oligomers regardless of their frequency and thdr position.
  • the sequence which is generated using the dgorithm described below is of high fidelity.
  • the dgorithm tolerates fdse positive signds from the hybridization dots as is indicated from the feet the sequence generated from the 105 hybridization vdues, which induded four less reliable vdues, was co ⁇ ect.
  • This fiddity in sequencing by hybridization is due to the "all or none" kinetics of short oligonucleotide hybridization and the difference in duplex stability that exists between perfectly matched duplexes and mismatched duplexes.
  • the ratio of duplex stability of matched and end-mismatched duplexes increases w h decreasing duplex length.
  • Image files are andyzed by an image andysis program, like DOTS program (D ⁇ nanac et al, 1993), and scded and evduated by statisticd functions included, e.g., in SCORES program (Drmanac etal. 1994). From the distribution of the signds an optimd threshold is determined for transforming signd into +/- output. From the position of the labd detected, F + P niicleotide sequences from the fragments wodd be determined by combining the known sequences of the immobilized and labded probes corresponding to the labeled positions.
  • the complete nudeic add sequence or sequence subfragments of the original molecule such as a human chromosome, would then be assembled from the overlapping F + P sequence determined by computationd deduction.
  • One option is to transform hybridization signds e.g., scores, into +/- output during the sequence assembly process. In this case, assembly will start with a F + P sequence with a very high score, for example F + P sequence AAAAAATTTTTT . Scores of dl four possible overlapping probes AAAAATTTTTTA , AAAAA1 I ' l l 1 1 , AAAAATTTTTTC and AAAAATTTTTTG and tiiree additional probes that are different at the beginning (TAAAAATTTTTT, ;
  • CAAAAATTTTTT, ; GAAAAATTTTTT, are compared and three outcomes defined: (i) only the starting probe and only one of the four overlapping proves have scores that are significantly positive relatively to the other six probes, in this case the AAAAAATTTTTT sequence will be extended for one nudeotide to the right; (ii) no one probe except the starting probe has a significantly positive score, assembly will stop, e.g., the AAAAAATTTTT sequence is at the end of the DNA molecde that is sequenced; (iii) more than one significantly positive probe among the overlapped and or other three probes is found; assembly is stopped because of the error or branching (Drmanac etal, 1989).
  • the present inventor particularly contemplates that hybridization is to be carried out for up to several hours in high sdt concentrations at a low temperature (-2°C to 5°C) because of a rdatively low concentration of target DNA that can be provided.
  • SSC buffer is used instead of sodium phosphate buffer (Drmanac et al, 1990), which predpitates at 10°C. Washing does not have to be extensive (a few minutes) because of the second step, and can be completely eliminated when the hybridization cyding is used for the sequencing of highly complex DNA samples.
  • the same buffer is used for hybridization and washing steps to be able to continue with the second hybridization step with labded probes.
  • each array e.g., a 8 x 8 mm array
  • one labeled, probe e.g., a 6-mer
  • a 96-tip or 96-pin device wodd be used, performing this in 42 operations. Again, a range of discriminatory conditions could be employed, as previously described in the sdentific literature.
  • the present inventor particularly contemplates the use of the following conditions. First, after adding labded probes and incubating for several minutes only (because of the high concentration of added oligonudeotides) at a low temperature (0-5°C the temperature is increased to 3-10°C depending on F + P length, and the washing buffer is added. At this time, the washing buffer used is one compatible with any ligation reaction (e.g., 100 mM salt concentration range). After adding ligase, the temperate is increased again to 15-37°C to dlow fast ligation (less than 30 min) and further discriinination of full match and mismatch hybrids.
  • cationic detergents are dso contemplated for use in Format 3 SBH, as described by Pontius & Berg (1991 , incorporated herein by reference). These authors describe the use of two simple cationic detergents, dodecy- and cetyltrimethylammonium bromide (DTAB and CTAB) in DNA renaturation.
  • DTAB and CTAB dodecy- and cetyltrimethylammonium bromide
  • DTAB and CTAB are variants of the quaternary amine tetramethylammonium bromide (TMAB) in which one of the methyl groups is replaced by dther a 12-carbon (DTAB) or a 16-carbon (CTAB) alkyl group.
  • TMAB is the bromide salt of the tetramethylammonium ion, a reagent used in nuddc acid renaturation experiments to decrease the G-C content bias of the melting temperature.
  • DTAB and CTAB are similar in structure to sodium dodecyl sdfate (SDS), with the leplacement of the negativdy charged sulfate of SDS by a positively charged quaternary amine. While SDS is commody used in hybridization buffers to reduce nonspecific binding and inhibit nucl ⁇ ases, it does not greatly affect the rate of renaturation.
  • SDS sodium dodecyl sdfate
  • ligase technology is wdl established within the field of molecular biology.
  • Hood and colleagues described a ligase-mediated gene detection technique (Landegren et al, 1988), the methodology of which can be readily adapted for use in Format 3 SBH. Wu &
  • Wdlace dso describe the use of bacteriophage T4 DNA ligase to join two adjacent, short synthetic olignucleotides.
  • Their oligo ligation reactions were carried out in 50 mM Tris HC1 pH 7.6, 10 mM MgCl 2 , 1 mM ATP, 1 mM DTT, and 5% PEG. Ligation reactions were heated to 100 Q C for 5-10 min followed by cooling to 0°C prior to the addition of T4 DNA ligase (1 unit; Bethesda Research Laboratory). Most ligation reactions were carried out at 30°C and terminated by heating to 100°C for 5 min.
  • phosphor storage screen technology and Phosphorlmager as a scanner may be used (Molecdar Dynamics, Sunnyvde, CA). Chips are put in a cassette and covered by a phosphorous screen. After 1 -4 hours of exposure, the screen is scanned and the image file stored a: a computer hard disc.
  • CCD cameras and epifluorescent or confocal microscopy are used.
  • detection can be perfb ⁇ ried as described by Eggers et al (1994, incorporated herein by reference).
  • Charge-coupled device (CCD) detectors serve as active solid supports that quantitativdy detect and image the distribution of labeled target molecules in probe-based assays, These devices use the inherent characteristics of microdectronics that accommodate highly parallel assays, ultrasensitive detection, high throughput, integrated data acquisition and computation. Eggers ei al. (1994) describe CCDs for use with probe-based assays, such as Format 3 SBH of the present invention, that dlow quantitative assessment within seconds due to the high sensitivity and direct coupling employed.
  • the integrated CCD detection approach enables the detection of molecdar binding events on chips.
  • the detector rapidly generates a two-dimensiond pattern that umqudy characterizes the sample.
  • distinct biological probes are immobilized directly on the pixds of a CCD or can be attached to a disposable cover slip placed on the CCD surface.
  • the sample molecules can be labeled with radioisotope, che ⁇ tiluminescent or fluorescent tags.
  • photons or radioisotope decay products are emitted at the pixel locations where the sample has bound, in the case of Format 3, to two complementary probes.
  • dectron-hole pdrs are generated in the silicon when the charged particles, or radiation from the labded sample, are incident on the CCD gates. Electrons are then collected beneath adjacent CCD gates and sequentially read out on a display modde. The number of photodectrons generated at each pixel is directly proportiond to the number of molecdar binding events in such proximity. Consequently, molecular binding can be quantitativdy determined (Eggers el al, 1994).
  • the collection efficiency is improved by a factor of at least 10 over lens-based techniques such as those found in conventiond CCD cameras. That is, the sample (emitter) is in near contact with the detector (imaging a ⁇ ay), and this eliminates conventiond imaging optics such as lenses and mirrors.
  • radioisotopes When radioisotopes are attached as reporter groups to the target molecdes, energetic particles are detected.
  • Several reporter groups that emit particles of varying energies have been successfully utilized with the micro-fabricated detectors, including 32 P, ⁇ P, 35 S, ,4 C and 125 L.
  • the higher eneTgy particles, such as from 32 P provide the highest molecdar detection sensitivity, whereas the lower energy particles, such as from ⁇ S, provide better resolution.
  • the choice of the radioisotope reporter can be tailored as required.
  • the detection performance can be predicted by cdcdating the signd-to-noise ration (SNR), as described by Eggers et al (1994).
  • An dtemative luminescent detection procedure involves the use of fluorescent or chemiluminescent reporter groups attached to the target molecdes.
  • the fluorescent labels can be attached covalently or through interactioa Fluorescent dyes, such as ethidium bromide, with intense absorption bands in the near UV (300-350 nm) range and principd emission bands in the visible (500-650 nm) range, axe most suited for the CCD devices employed since the quantum efficiency is several orders of magnitude lower at the excitation wavdength then at the fluorescent signal wavelength.
  • the polysilicon CCD gates have the bdlt-in caparity to filter away the contribution of incident light in the UV range, yet are very sensitive to the visible luminescence generated by the fluorescent reporter groups,
  • Such inherently large discrimination agdnst UV excitation enables large SNRs (greater than 100) to be achieved by the CCDs as formulated in the incorporated paper by Eggers et al (1994).
  • hybridization matrices may be produced on inexpensive SiOj wafers, which are subsequently placed on the surface of the CCD fdlowing hybridization and drying. This format is economicdly eff ⁇ rient since the hybridization of the DNA is conducted on inexpensive disposable Si0 2 wafers, thus dlowing reuse of the more expensive CCD detector. Altemativdy, the probes can be immobilized directly on the CCD to create a dedicated probe matrix. To immobilize probes upon the Si0 2 coating, a uniform epoxide layer is linked to the film surface, employing an epoxy-silane reagent and standard Si0 2 modification chemistry.
  • Amine-modified oligonudeotide probes are then linked to the Si0 2 surface by means of secondary amine formation with the epoxide ring.
  • the resulting linkage provides 17 rotatable bonds of separation between the 3 base of the oligonudeotide and the Si0 2 surface.
  • the reaction is performed in 0.1 M KOH and incubated at 37°C for 6 hours.
  • Cycling hybridizations are one possible method for increasing the hybridization signd. In one cycle, most of the fixed probes will hybridize with DNA fragments with tail sequences non-complementary for labeled probes. By increasing the temperature, those hybrids will be mdted. In the next cycle, some of them (-0.1%) will hybridize with an appropriate DNA fragment and additiond labded probes will be ligated. In this case, there occurs a discriminative melting of DNA hybrids with mismatches for both probe sets simdtaneously.
  • dl components are added before the cyding starts, at the 37°C for T4, or a higher temperature for a thermostable ligase. Then the temperature is decreased to 15-37°C and the chip is incubated for up to 10 minutes, and then the temperature is increased to 37°C or higher for a few minutes and then again reduced. Cycles can be repeated up to 10 times. In one variant, an optimal higher temperature (10-50°C) can be used without cycling and longer ligation reaction can be performed (1-3 hours).
  • the procedure described herein dlows complex chip manufacturing using standard synthesis and pre se spotting of oligonucleotides because a relatively smdl number of oligonucleotides are necessary. For example, if dl 7-mer oligos are synthesized (16384 probes), lists of 256 million 14-mers can be ddesmined.
  • One important variant of the invented method is to use more than one differently labeled probe per base array. This can be executed with two purposes in mind; mdtiplexing to reduce number of s ⁇ paratdy hybridized arrays; or to determine a list of even longer oligosequences such as 3 x 6 or 3 x 7. In this case, if two labds are used, the specifidty of the 3 consecutive oligonucleotides can be dmost absolute because positive sites must have enough signals of both labds.
  • a further and additiond variant is to use chips containing BxNy probes with y being from 1 to 4 . Those chips dlow sequence reading in different frames. This can dso be achieved by using appropriate sets of labded probes or both F and P probes codd have some unspecified end positions (i.e., some element of termind degeneracy). Universal bases may dso be employed as part of a linker to join the probes of defined sequence to the solid support. This makes the probe more avdlable to hybridization and makes the construct more stable. If a probe has 5 bases, one may, e.g., use 3 universd bases as a linker (FIG.4). EXAMPLE It?
  • Sequence assembly may be inte ⁇ upted where ever a given overlapping (N-l ) mer is duplicated two or more times. Then either of the two N-mers differing in the last nudeotide may be used in extending the sequence. This branching point limits unambiguous assembly of sequence. Reassembling the sequence of known oligonucleotides that hybridize to the target nucldc add to generate the complete sequence of the target nuddc acid may not be accomplished in some cases. This is because some information may be lost if the target nudeic acid is not in fragments of appropriate size in relation to the size of oligonudeotide that is used for hybridizing. The quantity of information lost is proportiond to the length of a target being sequenced. However, if sufficiently short targets are used, their sequence msy be unambiguously determined.
  • sequence subfragment results if any part of the sequence of a target nudeic acid starts and ends with an (N-l)mer that is repeated two or more times within the target sequence.
  • sequence subfragments are sequences generated between two points of branching in the process of assembly of the sequences in the method of the invention.
  • the sum of dl subfragments is longer than the actud target nudeic acid because of overlapping short ends.
  • subfragments may not be assembled in a linear order without additiond information since they have shared (N-l)mers at their ends and starts.
  • Different numbers of subfragments axe obtained for each nucleic add target depending on the number of its repeated (N-l) m ⁇ rs. The number depends on the vdue of N-l and the length of the target.
  • P (K, L r ) represents the probability of an N-mer occurring K-times on a fiagment L f base long.
  • P (K, L r ) represents the probability of an N-mer occurring K-times on a fiagment L f base long.
  • a representative library with sufficiently sho ⁇ inserts of target nucldc arid may be used.
  • inserts it is possible to reconstruct the individual target by the method of the invention.
  • the entire sequence of a large nucldc acid is then obtained by overlapping of the defined individud insert sequences.
  • This example describes an dgorithm for generation of a long sequence written in a four letter dphabet from constituent k-tuple words in a n ⁇ iimd number of separate, randomly defined fragments of a starting nudeic acid sequence where K is the length of an oligonudeotide probe.
  • the algorithm is primarily intended for use in the sequencing by hybridization (SBH) process.
  • the dgorithm is based on subfiagments (SF), informative fragments (IF) and the possibility of using pools of physicd nuddc sequences for defining informative fragments.
  • subfiagments may be caused by branch points in the assembly process resdting from the repetition of a K-l oligomer sequence in a target nuddc acid.
  • Subfragments are sequence fragments found between any two repetitive words of the length K-l that occur in a sequence. Mdtiple occurrences of K-l words are the cause of interruption of ordering the overlap of K-words in the process of sequence generation. Interruption leads to a sequence remaining in the form of subfiagments. Thus, the unambiguous segments between branching points whose order is not uniquely determined are cdled sequence subfragments.
  • Informative fragments are defined as fragments of a sequence that are determined by the nearest ends of overlapped physicd sequence fragments.
  • a certain number of physical fragments may be pooled without losing the possibility of defining informative fragments.
  • the total length of randomly pooled fragments depends on the length of k-tuples that are used in the sequencing process.
  • the dgorithm consists of two main units. The first part is used for generation of subfragments from the set of k-tuples contained in a sequence. Subfiagments may be generated within the coding region of physicd nucldc acid sequence of certain sizes, or within the informative fragments defined within long nucleic acid sequences. Both types of fragments are members of the basic library. This dgorithm does not describe the determination of the content of the k-tuples of the informative fragments of the basic library, i.e. the step of preparation of informative fragments to be used in the sequence generation process.
  • the second part of the dgorithm determines the linear order of obtained subfragments with the memepose of regenerating the complete sequence of the nucleic acid fragments of the basic library.
  • a second, ordering library is used, made of randomly pooled fragments of the starting sequence.
  • the dgorithm does not include the step of combining sequences of basic fragments to regenerate an entire, egabase plus sequence. This may be accomplished using the link-up of fragments of the basic library which is a prerequisite for informative fragment generation. Altemativdy, it may be accomplished after generation of sequences of fragments of the basic library by this dgorithm, using search for their overlap, based on the presence of common end-sequences.
  • the dgorithm requires ndther knowledge of the number of appearances of a given k-tuple in a nucldc acid sequence of the basic and ordering libraries, nor does it require the information of which k-tuple words are present on the ends of a fiagment.
  • the dgorithm operates with the mixed content of k-tuples of various length.
  • the concept of the dgorithm enables operations with the k-tuple sets that contain fdse positive and felse negative k- tuples. Ody in specific cases does the content of the f se k-tuples primarily influence the completeness and correctness of the generated sequence.
  • the dgorithm may be used for optimization of parameters in simulation experiments, as well as for sequence generation in the actud SBH experiments e.g. generation of the genomic DNA sequence.
  • sequence generation in the actud SBH experiments e.g. generation of the genomic DNA sequence.
  • the choice of the oligonucleotide probes (k-tuples) for practical and convenient fragments and/or the choice of the optimd lengths and the number of fragments for the defined probes are espeddly important
  • This part of the dgorithm has a central role in the process of the generation of the sequence from the content of k-tuples. It is based on the udque ordering of k «tuples by means of maximal overlap.
  • the main obstacles in sequence generation are specific repeated sequences and fdse positive and or negative k-tuples.
  • the aim of this part of the dgorithm is to obtain the minimd number of the longest possible subfragments, with correct sequence.
  • This part of the dgorithm consists of one basic, and several control steps. A two-stage process is necessary since certain information can be used only after generation of dl primary subfiagments.
  • the main problem of sequence generation is obtaining a repeated sequence from word contents that by definition do not carry information on the number of occurrences of the particdar k-tuples.
  • the concept of the entire dgorithm depends on the basis on which this problem is solved.
  • pSFs contain an excess of sequences and in the second case, they contain a deficit of sequences.
  • the first approach requires elimination of the excess sequences generated, and the second requires permitting ⁇ ltiple use of some of the subfragments in the process of the find assembling of the sequence.
  • k-tuple X is unambiguously maxi ⁇ idly overlapped with k-tuple Y if and only if, the rightmost k-l end of k-tuple X is present ody on the leftmost end of k-tuple Y.
  • This rule dlows the generation of repetitive sequences and the formation of surplus sequences.
  • k-tuple X is unambiguously maximdly overlapped with k-tuple Y if and ody if, the rightmost K- 1 end of k-tuple X is present only on the leftmost end of k-tuple Y and if the leftmost K-l end of k- tuple Y is not present on the rightmost end of any other k-tuple.
  • the dgorithm based on the stricter rule is simpler, and is described herein.
  • the process of dongation of a given subfragment is stopped when the right k-l end of the last k-tuple included is not present on the left end of any k-tuple or is present on two or more k-tuples. If it is present on ody one k-tuple the second part of the rule is tested. If in addition there is a k-tuple which differs from the previously included one, the assembly of the given subfragment is terminated ody on the first leftmost position. If this additiond k-tuple does not exist, the conditions are met for unique k-l overlap and a given subfragment is extended to the right by one element.
  • a supplementary one is used to dlowthe usage of k-tuples of different lengths.
  • the maximd overlap is the length of k-l of the shorter k-tuple of the overlapping pair.
  • Generation of the pSFs is performed starting from the first k-tuple from the file in • which k-tuples are displayed randomly and independently from their order in a nudeic add sequence.
  • the first k-tuple in the file is not necessarily on the beginning of the sequence, nor on the start of the particular subfragment
  • the process of subfragment generation is performed by ordering the k-tuples by means of unique overlap, which is defined by the described wle. Each used k-tuple is erased from the file.
  • the bdlding of subfragment is terminated and the bmldup of another pSF is started. Since generation of a majority of subfragments does not begin from thrir actud starts, the formed pSF are added to the k-tuple file and are considered as a longer k-tuple. Another possibility is to form subfragments going in both directions from the starting k- tuple. The process ends when further overlap, i.e. the extension of any of the subfiagments, is not possible.
  • the pSFs can be divided in three groups: 1 ) Subfiagments of the maximd length and correct sequence in cases of exact k-tuple set; 2) short subfiagments, formed due to the used of the maximd and unambiguous overlap rule on the incomplete set, and/or the set with some false positive k-tuples; and 3) pSFs of an inco ⁇ ect sequence-
  • the incompleteness of the set in 2) is caused by fdse negative resdts of a hybridization experiment, as wdl as by using an inco ⁇ ect set of k-tuples.
  • fdse positive and false negative k ' tuples can be : a) misconnected subfiagments; b) subfragments with the wrong end and c) false positive k-tuples which appears as fdse minimd subfragments.
  • pSFs are generated because of the impossibility of maximd overlapping.
  • pSFs are generated because of the impossibility of unambiguous overlapping.
  • the process of co ⁇ ecting subfiagments with e ⁇ ors in sequence and the linking of unambiguously connected pSF is performed after subfragment generation and in the process of subfragment ordering.
  • the first step which consists of cutting the misconnected pSFs and obtaining the find subfragments by unambiguous connection of pSFs is described below.
  • Recognition of the misconnected subfragments is more strictly defined when a repeated sequence does not appear at the end of the fiagment. In this situation, one can detect further two subfragments, one of which contains on its leftmost, and the other on its rightmost end k-2 sequences which are dso present in the misconnected subfragment. When the repeated sequence is on the end of the fragment, there is ody one subfragment which contains k-2 sequence causing the mistake in subfragment formation on its leftmost or rightmost end.
  • the removd of misconnected subframents by their cutting is performed according to the common rule: If the leftmost or rightmost sequence of the length of k-2 of any subfragments is present in any other subfragment, the subfragment is to be cut into two subfragments, each of them containing k-2 sequence. This nile does not cover rarer situations of a repeated end when there are more than one fdse negative k-tuple on the point of repeated k-l sequence.
  • Misconnected subfiagments of this kind can be recognized by using the information from the overlapped fragments, or informative fragments of both the basic and ordering libraries,
  • the misconnected subfragment will remain when two or more felse negative k-tuples occux on both positions which contain the identicd k-l sequence. This is a very rare situation since it requires at least 4 spedfic false k-tuples.
  • An additiond nile can be introduced to cut these subfragments on sequences of length k if the given sequence can be obtained by combination of sequences shorter than k-2 from the end of one subfragment and the start of another.
  • a fiagment beside at least two identicd k-l sequences, contains any k-2 sequence from k-l or a fiagment contains k-2 sequence repeated at least twice and at least one false negative k-tuple containing given k-2 sequence in the middle, etc.
  • the aim of this part of the dgorithm is to reduce the number of pSFs to a minimd number of longer subfiagments with correct sequence.
  • the generation of unique longer subfragments or a complete sequence is possible in two situations.
  • the first situation concerns the specific order of repeated k-I words.
  • Some or dl maximdly extended pSFs (the first group of pSFs) can be uniquely ordered.
  • S and E are the start and end of a fragment
  • a, b , and c are different sequences specific to respective subfragments
  • Rl and R2 are two k-l sequences that are tandemly repeated
  • five subfragments are generated (S-Rl , Rl-a-R2, R2-b-Rl, R1-C-R2, and R-E).
  • the process of ordering of subfragments is similar to the process of their generation. If one considers subfragments as longer k-tuples, ordering is performed by their unambiguous connection via overlapping ends.
  • the informationd basis for unambiguous connection is the division of subfiagments generated in fragments of the basic library into groups representing segments of those fragments.
  • the method is andogous to the biochemicd solution of this problem based on hybridization with longer oligonudeotides with relevant connecting sequence.
  • the connecting sequences are generated as subfragments using the k-tuple sets of the appropriate segments of basic library fragments.
  • Rdevant segments are defined by the fragments of the ordering library that overlap with the respective fragments of the basic library.
  • the shortest segments are informative fragments of the ordering library.
  • RNAse treatment may utilize RNAse A an endoribonudease that specifically attacks single-stranded RNA 3 to pyrimidine residues and cleaves the phosphate linkage to the adjacent nudeotide.
  • the end products are pyrimidine 3 phosphates and oligonucleotides with termind pyrimidine 3 phosphates.
  • RNAse A works in the absence of cofactors and divdent cations. To utilize an RNAse, one would generally incubate the chip in any appropriate
  • One approach is to sdect a probe that extends the positively hybridized probe TGCAAA for one nucleotide to the right, and which extends the probe TATTCC one nudeotide to the left.
  • these 8 probes GCAAAA, GCAAAT, GCAAAC, GCAAAG and ATATTC, TTATTC, CTATTC, GTATTC
  • two questionable nucleotides are determined.
  • DNA fragments as long as 5-20 kb may be sequenced without subdoning.
  • the speed of sequencing readily may be about 10 million bp/day/hybridization instrument. This performance dlows for resequenci ⁇ g a large fraction of human genes or the human genome repeatedly from scientificdly or medically interesting individuds. To resequence 50% of the human genes, about 100 million bp is checked. That may be done in a relativdy short period of time at an affordable cost.
  • mRNAs which may be converted into cDNAs
  • genomic DNA from particdar tissues or genomic DNA of patients with particular disorders may be used as starting materids.
  • genes or genomic fragments of appropriate length may be prepared either by cloning procedures or by in vitro amplification procedures (for example by PCR). If cloning is used, the minimd set of clones to be andyzed may be sdected from the libraries before sequencing.
  • Cloning may increase the amount of hybridization data about two times, but does not require tens of thousands of PCR primers.
  • SNUPs single nucleotide polymorphisms
  • SNUPs may be scored in every individud from relevant families or populations by amplifying markers and arraying them in the form of the a ⁇ ay of subarrays.
  • Subarrays contain the same marker amplified from the andyzed individuals.
  • For each marker as in the diagnostics of known mutations, a set of 6 or less probes positive for one dlde and 6 or less probes positive for the other dlele may be selected and scored. From the significant association of one or a group of the markers with the disorder, chromosomal position of the responsible gene(s) may be determined. Because of the high throughput and low cost, thousands of markers may be scored for thousands of individuds.
  • This amount of data dlows localization of a gene at a resolution level of less than one million bp as well as localization of genes involved in poJygenic diseases.
  • Localized genes may be identified by sequencing parti ⁇ dar regions from relevant normd and affected individuds to score a mutation(s).
  • PCR is prefe ⁇ ed for amplification of markers from genomic DNA
  • Each of the markers require a spedfic pair of primers.
  • the existing markers may be convertible or new markers may be defined which may be prepared by cutting genomic DNA by Hga 1 type restriction enzymes, and by ligation with a pair of adapters.
  • EXAMPLE 22 Detection and Verification of Identity of DNA Fragments DNA fragments generated by restriction cutting, cloning ox in vitro amplification (e.g.
  • Identification may be performed by verifying the presence of a DNA band of specific size on gel electrophoresis.
  • a specific oligonucleotide may be prepared and used to verify a DNA sample in question by hybridization. The procedure devdoped here dlows for more effident identification of a large number of samples without preparing a specific oligonucleotide for each fragment.
  • a set of positive and negative probes may be sele ⁇ ed from the univeisd set for each fragment on the basis of the known sequences. Probes that are sdected to be positive usually are able to form one or a few overlapping groups and negative probes are spread over the whole inser
  • the amount of DNA may be estimated using intensities of the hybridization of several separate probes or one or more pools of probes. By comparing obtained intensities with intensities for control samples having • a known amount of DNA the quantity of DNA in dl spotted samples is determined simdtaneously. Because ody a few probes are necessary for identification of a DNA fiagment, and there are N possible probes that may be used for DNA N bases long, this application does not require a large set of probes to be sufficient for identification of any DNA segment. From one thousand 8-mers, on average about 30 full matching probes may be sele ⁇ ed for a 1000 bp fiagment. EXAMPLE 23
  • DNA fragments may be amplified from isolated viruses from up to 64 patients and resequenced by the described procedure. On the basis of the obtained sequence the optimd therapy may be selected. If there is a mixture of two virus types of which one has the basic sequence
  • the mutant may be identified by quantitative comparisons of its hybridization scores with scores of other samples, especidly control samples containing the basic vims type ody, Scores twice as smdl may be obtained for three to four probes that cover the site mutated in one of the two virus types present in the sample (see above).
  • EXAMPLE 24 . , forensic apd Parental Identification
  • Sequence polymorphisms make an individud genomic DNA unique. This permits analysis of blood or other body Adds or tissues from a crime scene and comparison with samples from crimind suspects. A sufficient number of polymorphic sites are scored to produce a unique signature of a sample. SBH may easily score single nudeotide polymoiphisms to produce such signatures,.
  • DNA may be prepared and polymorphic lod amplified from the child and adults; patterns of A or B dleles may be determined by hybridization for each. Comparisons of the obtained patterns, dong with positive and negative controls, dde in the deteimination of familid rdationships. In this case, only a significant portion of the alldes need match with one parent for identification. Large numbers of scored loci dlow for the avoidance of statistical errors in the procedure or of masking effects of de nov ⁇ mutations. 6567
  • DNA may be prepared from the environment and particdar genes amplified using primers co ⁇ esponding to conservative sequences, DNA fiagments may be cloned preferentidly in a plasmid ve ⁇ or (or diluted to the level of one molecde per well in mdtiwdl plates and than amplified in vitro). Clones prepared this way may be res ⁇ quenced as described above. Two types of information are obtained.
  • a catalogue of different species may be defined as well as the density of the individuds for each species. Another segment of information may be used to measure the influence of ecologicald fa ⁇ ors or pollution on the ecosystem. It may reved whether some species are eradicated or whether the abundance ratios among species is dtered due to the pollution.
  • the method dso is applicable for sequencing DNAs from fossils.
  • EXAMPLE 26 petectiop or Quantification of Nucleic Arid Species DNA or RNA species may be detected and quantified by employing a probe pair induding an unlabeled probe fixed to a substrate and a labded probe in a solution.
  • udigated labded probe indicates the presence of a sample species while the quantity of label indicates the expression level of the species
  • An infectious agent with about 1 Okb or more of DNA may be dete ⁇ ed using a support-bound detection chip without the use of polymerase chain reaction (PCR) or other target amplification procedures.
  • PCR polymerase chain reaction
  • the genomes of infectious agents induding ba ⁇ eria and viruses are assayed by amplification of a single target nudeotide sequence through PCR and detection of the presence of target by hybridization of a labdled probe specific for the target sequence. Because such an assay is specific for ody a single target sequence it therefore is necessary to amplify the gene by methods such as PCR to provide sufficient target to provide a detectable signd.
  • Such multiple probes may be of overlapping sequences of the target nudeotide sequence but may dso be non-overlapping sequences as well as non-adjacent Such probes preferably have a length of about 5 to 12 nucleotides.
  • an a ⁇ ay or super array may be prepared which consists of a complete set of probes, for example 40966-mer probes.
  • Arrays of this type are universd in a sense that they can be used for detection or pneumonia to complete sequencing of any nucleic acid species.
  • Individud spots in an a ⁇ ay may contain single probe species or mixtures of probes, for example N(l-3) B(4-6) N(l-3) type of mixtures that are synthesized in the single reaction (N represents dl four nucleotides, B one specific nudeotide and where the associated numbers are a range of numbers of bases le., 1-3 means "from one to three bases”.)
  • the universal set of probes may be subdivided in many subsets which are spotted as unit arrays separated by barriers that prevent spreading of hybridization buffer with sample and labeled probe(s).
  • For detection of a nucleic acid species with a known sequence one of more oligonucleotide sequences comprising both udabelled fixed and labded probes in solution may be sdected.
  • Labeled probes are synthesized or selected from the presynthesized complete sets of, for example, 7-mers.
  • the labded probes are added to co ⁇ esponding unit arrays of fixed probes such that a pdr of fixed and labeled probes will adjacently hybridize to the target sequence such that upon administration of ligase the probes will be covdently bound.
  • the subarrays in the a ⁇ ay were partitioned from each other by physicd barriers, e.g., a hydrophobic strip, that dlowed each subarray to be hybridized to a sample without cross- contamination from adjacent subarrays.
  • the hydrophobic strip is made from a solution of silicone (e.g., household silicone glue and sed paste) in an appropriate solvent (such solvents are well known in the art). This solution of silicone grease is applied between the subarrays to form lines which after the solvent evaporates a ⁇ as hydrophobic strips separating the cells.
  • the free 5-mers and the bound 5-mers are combined to produce dl possible 10-mers for sequencing a known DNA sequence of less than 20 kb.
  • 20 kb of double stranded DNA is denatured into 40 kb of single-stranded DNA.
  • This 40 kb of ss DNA hybridizes to about 4% of dl possible 10-mers.
  • This low frequency of 10- er binding and the known target sequence dlow the pooling of free or solution (nonbound) 5- mers for treatment of each suba ⁇ ay, without a loss of sequence information.
  • the target DNA in this embodiment represents two-600 bp segments of the HTV vims. These 600 bp segments axe represented by pools of 60 overlapping 30-mers (the 30-mers overlap each adjacent 30 mer by 20 nudeotides). The pools of 30-mers mimic a target DNA that has been treated using techniques wdl known in the art to shear, digest and/or random PCR the target DNA " to produce a random pool of very smdl fragments. 6567
  • the free 5-mers are labeled with radioactive isotopes, biotin, fluorescent dyes, dc.
  • the labded free 5-mers are then hybridized dong with the bound 5-mers to the target DNA and ligated.
  • 300-1000 units of ligase are added to the reaction.
  • the hybridization conditions were worked out following the teachings of the previous examples. Following ligation and removd of the target DNA and excess free probe, the array is assayed to determine the location of labded probes (uiing the techniques described in the examples above).
  • the overlapping sequence of the bound 5-mers in these ten new dots identifies which free, labded 5-mer is bound in each new dot.
  • repetitive DNA sequences in the target DNA are sequenced with "spacer oligonucleotides" in a modified Format III approach.
  • Spacer oligonudeotides of varying lengths of the repetitive DNA sequence (the repeating sequence is identified on a first SBH run) axe hybridized to the target DNA dong with a first known adjoining oligonucleotide and a second kn ⁇ wn, or group of possible oligonudeotides adjoining the other side of the spacer (known from the first SBH run).
  • the two adjacent oligonudeotides can be ligated to the spacer, If the first known oligonucleotide is fixed to a substrate, and the second known or possible oligonudeotide(s) is labded, a bound ligation produ ⁇ including the labded second known or possible oligonucl ⁇ otide(s) is formed when a spacer of the proper length is hybridized to the target DNA.
  • branch points in the target DNA are sequenced using a third set of oligonucleotides and a modified Format UI approach. After a first SBH run, several brandi points may be identified when the sequence is compiled. These can be solved by hybridizing oligonucleotide(s) that overlap partlyly with one of the known sequences leading into the branch point and then hybridizing to the target an additiond oligonudeotide that is labded and corresponds to one of the sequences that comes out of the branch point When the proper oligonucleotides are hybridized to the target DNA, the labeled oligonudeotide can be ligated to the other(s).
  • a first oligonucleotide that is offset by one to several nucleotides from the branch point is selected (so that it reads into one of the branch sequences), a second oligonucleotide reading from the first and into the branch point sequence is also sde ⁇ ed, and a set of third oligonudeotides that co ⁇ espond to dl the possible branch sequences with an overlap of the branch point sequence by one or a few nucleotides (co ⁇ esponding to the first oligonudeotide) is sdected.
  • oligonucleotides are hybridized to the target DNA, and only the third oligonudeotide with the proper branch sequence (that matches the branch sequence of the first oligonudeotide) will produce a ligation produrt with the first and second oligonudeotides.
  • Muhiplexinp Probes for Analyzing a Target Nucleic Acid sets of probes are labeled with different labels so that each probe of a set can be differentiated from the other probes in the set.
  • the set of probes may be contacted with target nucleic acid in a single hybridization reaction without the loss of any probe information.
  • the different labels are different radioisotopes, or different flourescent labds, or different EMLs. These sets of probes may be used in dther Format I, Format II or Format III SBH.
  • the different labels are EMLs, which can be detected by electron capture mass spectrometry (EC-MS).
  • EMLs may be prepared from a variety of backbone molecules, with certain aromatic backbones being particularly prefe ⁇ ed, e.g., see Xu et ⁇ l, J. Chromatog. 764:95-102 (1997).
  • the EML is attached to a probe in a reversible and stable manner, and after the probe is hybridized to target nucldc arid, the EML is removed from the probe and identified by standard EC-MS (e.g., the EC-MS may be done by a gas chromatograph-mass spectrometer).
  • Format III SBH has sufficient discrimination power to identify a sequence that is present in a sample at 1 part to 99 parts of a similar sequence that differs by a single nucleotide.
  • Format Dl can be used to identify a nucldc arid present at a very low concentration in a sample of nucleic acids, e.g., a sample derived from blood.
  • the two sequences are for cystic fibrosis and the sequences differ from each other by a deletion of three nucleotides.
  • Probes for the two sequences were as follows, probes distinguishing the deletion from wild type were fixed to a substrate, and a labded contiguous probe was common to both. Using these targets and probes, the deletion mutant codd be detected with Format III SBH when it was present at one part to ninety nine parts of the wild-type. 6567
  • An apparatus for andyz g a nucleic acid can be constructed with two a ⁇ ays of nucleic adds, and an optiond materid that prevents the nucleic acids of the two arrays from mixing until such mixing is desired.
  • the arrays of the apparatus may be supported by a variety of substrates, induding but not limited to, nylon membranes, nitrocellulose membranes, or other materids disclosed above, In prefe ⁇ ed embodiments, one of the substrate is a membrane separated into sectors by hydrophobic strips, cor a suitable support materid with wdls which may contain a gd or sponge.
  • probes are placed on a sertor of the membrane, or in the well, the gel, or sponge, and a solution (with or without target nucldc acids) is added to the membrane or wdl so that the probes are solubilized. The sdution with the solubilized probes is then dlowed to contact the second array of nudeic acids.
  • the nucleic acids may be, but are not limited to, oligonudeotide probes, or target nucleic acids, and the probes or target nucldc adds may be labded.
  • the nucldc adds may be labded with any labds conventiondly used in the art, including but not limited to radioisotopes, fluorescent labds o ⁇ dectrophore mass labds.
  • the materid which prevents mixing of the nucldc adds may be disposed between the two a ⁇ ays in such a way that when the materid is removed the nucleic acids of the two a ⁇ ays mix together.
  • This materid may be in the form of a sheet, membrane, or other barrier, and this material may be comprised of any materid that prevents the mixing of the nudeic acids.
  • This apparatus may be used in Format I SBH as follows: a first array of the apparatus has target nudeic acids that are fixed to the substrate, and a second array of the apparatus has nucleic add probes that are labeled and can be removed to interrogate the target nudeic acid of the first array.
  • the two arrays are optiondly separated by a sheet of materid that prevents the probes from contacting the target nudeic acid, and when this sheet is removed the probes can inte ⁇ ogate the target.
  • the array of targets may be "read" to determine which probes formed perfe ⁇ matches with the target. This reading may be automated or can be done raanudly (e.g., by eye with an autoradiogram).
  • Format U SBH the procedure followed would be similar to that described above except that the target is labded and the probes are fixed.
  • the apparatus may be used in Format III SBH as follows: two arrays of nuddc acid probes are formed, the nucleic arid probes of either or both arrays may be labded, and one of the arrays may be fixed to its substrate. The two arrays are separated by a sheet of material that prevents the probes from mixing.
  • a Format H reaction is initiated by adding target nucldc arid and removing the sheet dlowing the probes to mix with each other and the target Probes which bind to adjacent sites on the targrt are bound together (e.g., by base-stacking interactions or by covdently joining the backbones), and the resdts are read to determine which probes bound to the target at adjacent sites.
  • the fixed array can be read to determine which probes from the other array are bound together with the fixed probes. As with the above method, this reading may be automated (e.g., with an ELISA reader) or can be done manudly ( ⁇ .g., by eye with an autoradiogram).
  • the oligonudeotide probes are fixed in a three-dimensiond anay.
  • the three-dimensiond array is comprised of multiple layers, such that each layer may be andyzed separate and apart from the other layers, or all the layers of the three-dimensiond a ⁇ ay may be simultaneously analyzed.
  • Three dimensional a ⁇ ays indude for example, an a ⁇ ay disposed on a substrate having multiple depressions with probes located at different depths within the depressions (each level is made up of probes at similar depths within the depression); or an array disposed on a substrate having depressions of different depths with the probes located at the bottom of the depression, at the peaks separating the depressions or some combination of peaks and depressions (each level is made up of dl probes at a certain depth); or an array disposed on a substrate comprised of multiple sheets that are layered to form a three-dimensiond a ⁇ ay,
  • Materids for synthesizing these three-dimensiond a ⁇ ays are wdl known in the an, and indude the materids previously rerited in this specification as suitable as supports for probe arrays.
  • other suitable materids which can support oligonucleotide probes, and which preferably, are flexible may be used as substrates.
  • a plurality of distinct nucldc arid sequences were obtained from cDNA library, using Standard per, SBH sequence signature andysis and Sanger sequencing techniques.
  • the inserts of the library were amplified with per using primers specific for vector sequences which flank the inserts. These samples were spotted onto nylon membranes and interrogated with sdtable number of oligonucleotide probes and the intensity of positive binding probes was measured giving sequence signatures.
  • the clones were clustered into groups of similar or identical sequence signatures, and single representative dones were selected from each group for gd sequencing.
  • the 5' sequence of the amplified inserts was then deduced using the reverse M13 sequencing primer in a typicd Sanger sequencing protocol.
  • an apparatus for mass producing arrays of probes may comprise a rotating drum or plate coupled with an ink-jet deposition apparatus, for example, a microdrop dosing head; and a suitable robotics systems, for example, an anorad gantry. A particdarly preferred embodiment of the apparatus will be described referring to Figs. 1-3.
  • the apparatus comprises a cylinder (1 ) to which a suitable substrate is fixed.
  • the substrate may be any of the materials previously described as suitable for an array of probes.
  • the substrate is a flexible material, and the a ⁇ ays are made dirertly on the substrate.
  • a flexible substrate is fixed to the cylinder and individud chips are fixed on the substrate. The a ⁇ ays are then made on each individud chip.
  • physicd barriers are applied to the substrate or chip and define an a ⁇ ay of wells.
  • the physicd barriers may be applied to the substrate or chip by the apparatus, or dternativdy, the physicd barriers are applied to the chips or substrate before they are fixed to the cylinder (I).
  • a single spot of oligonucleotide probes is then placed into each wdl, wherein the probes placed into an individud wdl may all have the same sequence, or the probes spotted into an individud well may have different sequences, ln a more prefe ⁇ ed embodiment the probe or probes spotted into each individud wdl in an array are different from the probe or probes spotted in 6567
  • Sequencing chips comprising dtiple arrays can then be assembled from these a ⁇ ays.
  • a motor (not shown) rotates the cylinder.
  • the cylinder's rotation speed is precisely determined by any of the ways wdl known in the art, including, for example using a fixed opticd sensor and light source that rotates with the cylinder.
  • a dispensing apparatus (3) moves along an arm (2) and can deliver probes or other reagents through a dispensing tip (8) to precise locations on the substrate or chips using the precise rotation speed cd ⁇ ilated above, by methods well known in the art.
  • the dispensing apparatus recdves probes or reagents from the reservoir (6) through the feeding line (7).
  • the reservoir (6) holds d] the necessary probes and other reagents for making the arrays.
  • the dispensing apparatus is depicted in Figure 3.
  • the dispensing apparatus may have one or multiple dispensing tips (14 & ⁇ 8).
  • Each dispensing tip has a sample wdl (13) in a body (12) that receives probes or other reagents through a sample line (10).
  • the pressure line (11) pressurizes the chamber (9) to a psi sufficient to force probes or reagents through the dispensing tip ( 14 &.8).
  • the sample line (10), wdl (13) and dispensing tip (14 & 8) must be flushed between each change in probe or reagent
  • An appropriate washing buffer is supplied through sample line (10) or through an optiond dedicated washing line (not shown) to the sample well (13) or optiondly a portion or dl of the chamber (9) may be filled with washing buffer.
  • the washing buffer is then removed from the sample wdl (13) and chamber (9) if necessary by an evacuation line (not shown) or through the sample line (10) and dispensing tip (14 & 8).
  • the substrate (with or without chips) is removed from the cylinder and a new substrate is fixed to the cylinder.
  • a target nucleic acid is interrogated with probes that are complexed (covdent or noncovde ⁇ t) to a plurality of discrete particles.
  • the discrete partides can be discriminated from each other based on a physicd property (or a combination of physical properties), and particles with differentiated by the physical property are complexed with different probes.
  • the probe is an oligonucleotide of a known sequence and 6567
  • a probe may be identified by the physicd property of the discrete particle.
  • Suitable probes for this embodiment include dl the probes that are described above in previous sections, including probes which are shorter in an informative sense than the probes full length.
  • the physicd property of the discrete particle may be any property, well known in the art, which allows particles to be differentiated into sets.
  • the partides could be differentiated into sets based on their size, flourescence, absorbance, dectromagnetic charge, or weight, or the particles could be labded with dyes, radionudides, ox EMLs.
  • Other suitable labds include ligands which can serve as specific binding members to a labeled antibody, chemiluminescers, enzymes, antibodies which can serve as a specific binding pair member for a labded ligand, and the like.
  • a wide variety of labds have been employed in immunoassays which can readily be employed.
  • Still other labds include antigens, groups with specific rea ⁇ ivity, and dectrochemically detectable moieties.
  • Still further labels include any of the labels recited above in previous sections. These labels and properties may be measured quantitativdy by methods wdl known in the art, induding for example, those methods described above in previous sections, and the partides may be differentiated on the basis of signd intensity or signd type (for one of the labels, e.g., different dye densities may be applied to a particle, or different types of dyes). In a preferred embodiment several physicd properties are combined and the different combinations of properties allow discrimination of the partides (e.g., ten sizes and ten colors could be combined to differentiate 100 pubert).
  • the pa tide-probes dlow the exploitation of standaid combinatorid approaches so that, for example, dl possible 10-mers can be synthesized using about 2000 reaction containers.
  • a first set of 1024 reactions are done to synthesize dl possible 5-mers on 1024 differcntidly labeled particles.
  • the resulting probe-particles are mixed together, and split into another set of 1024 reaction containers.
  • a second set of reactions are done with these samples to synthesize dl possible 5-mer extensions on the probes in the pools of partides.
  • the physicd property identifies the first five nucleotides of each probe and the reaction container will identify the identity of the second five nucleotides of every probe.
  • dl possible 10-mer probes axe synthesized using 2048 reaction containers, This approach is easily modified to make all possible n-mers for a large range of probe lengths.
  • the particles are separated into sets by the intensity of flourescence of the particles.
  • the particles in each set are prepared with varying densities of flourescent label, and thus, the particles have different flourescence intensities.
  • the flourescent intensity of flourescdn is rdated to concentration over a range of 1:300 to 1:300,000 (Loc hart et d., 1986), and between 1:3000 to 1:300,000 there is a linear relationship (so the fiourescein intensity is linear over a range of about 1-300).
  • 256 sets of particles are labeled with fiourescein (e.g., 3-259). 256 sets of partides allows all possible 4-mers to be attached to different sets of particles.
  • dl possible 5-mers can be made by having four pools of dl possible 4-mers and then extending the probes in each pool by A, G, C, or T.
  • all possible 6-mers can be made by having 16 pools of all possible 4-mers in which each pool of 4-mers is extended by one of the 1 possible two base permutations of A, G, C, and T (etc. for 7-mers there are 64 pools, 8-mers there are 256 pools, and so forth).
  • the 5-mer probes (in four pools) are used to inte ⁇ ogate a target nucldc acid.
  • the target nucleic acid is labeled with another flourescent dye, or other different label (as described above).
  • Labeled target is mixed with the four pools, and complementary probes in each pool hybridize to the target nucleic acid. These hybridization complexes are detected by methods wdl-known in the art, and the positively hybridizing probes are then identified by detecting the flourescence intensity of the particle.
  • the mixture of probe-particles and target nucleic arid are fed through a flow-cytometer or other separating instrument one particle at a time, and the particle label and the target are measured to determine which probes are complementary to the target nucleic add.
  • a set of free probes is labeled with another flourescent dye, or other label (as described above), and individud free probes are mixed with each pool of 5-mer probes (four pools) and then the mixtures are hybridized with the target nudeic arid.
  • An agent is added to covdently attach free probe to 5-mer probe (see previous sections for a description of suitable agents), when the free probe is bound to a site on the target nudeic arid that is adjacent to the site on which the 5-mer probe is bound (the free probe site must be adjacent the end of the 5- mer probe which can be ligated).
  • the particles are then assayed, by methods well known in the art, to determine which partides have been covdently coupled to the free probe, i.e., the particles which have the free probe Jabd, and the 5-mer probe is identified by the flourescent intensity of the 6567
  • the mixture of probe-partides, free probes, and t arget nudeic add are fed through a flow-cytometer one particle at a time, and the particle label and the free probe label are measured to determine which probes are complementary to the target nudeic acid.
  • a single apparatus houses all or most of the madpdations for an andysis of a target nucleic acid with the probe-particle complexes.
  • the apparatus has one or more reagent chambers in which buffer and labeled target nudeic acid are thoroughly mixed (target nucleic acid may be added manually or automaticdly).
  • the mixture is diquoted from the reagent chamber into a plurality of reaction chambers, and each reaction chamber has a pool of probe- particle complexes.
  • the probe-particles and target nucleic acid react under conditions which dlow complementary probes to bind with the target nudeic acid.
  • Excess target nudeic acid, i.e., nonbound is removed from the reaction chamber (e.g., by washing), and the particles bound to target nucldc acid are identified by the association of target nucleic acid label with the particles, and the probe is identified by the physicd property of the particle.
  • the partides move single file through a channel from the reaction chamber to the dete ⁇ ing device(s).
  • the particles are fractionated, for example, by size (e.g., exclusion chromatography), charge (e.g., ion exchange chromatography), and/or density-weight into thrir sets using one or a combinations of these physicd properties. These fractionated partides are then assayed by the dete ⁇ ing device(s).
  • the main reagent chamber is supplied with buffer, target nudeic arid, a pool of probe-partide complexes, and a chemicd or enzymatic ligating reagent. These components are thoroughly mixed and then diquoted from the reagent chamber into a plurality of reaction chambers. Each of the reaction chambers has a labded free probe.
  • the pool of probe particlese complexes may be placed in the reaction chamber with the free probe instead of adding them to the reagent chamber.
  • the free probes codd be added to the reagent chamber, and the pool of probe partides codd be added to the reaction chamber.
  • the probe-particles, target nudeic acid, and free probe react under conditions which allow free- and monkeye-probes to bind with adjacent sites on the target nucleic acid so that free 567 probe is ligated to the probe pub.
  • Excess free probe (i.e., nooligated), and target nucleic acid are removed from the reaction chamber (e.g., by washing), and the ligated probes are identified by the association of free probe Iabd with the partides, and the probes complexed to the paiticles are identified by the physicd property of the pukee.
  • the partides move single file through a channel from the reaction chamber to the dete ⁇ ing device(s). As single particles move past the dete ⁇ ing device(s) they measure free probe label covdently attached to the partides, and the physicd property of the particle.
  • the particles before or after removing excess probe and the target, are fractionated, for example, by size (e.g., exclusion chromatography), charge (e.g., ion exchange chromatography), and/or density weight into their sets using the physicd property.
  • size e.g., exclusion chromatography
  • charge e.g., ion exchange chromatography
  • density weight into their sets using the physicd property.
  • the pool of probe-particles are placed in the second reaction chamber.
  • Target and buffer are mixed in the reagent chamber and these are fed into the first reaction chamber which contains the labeled free probe.
  • the probe and target are mixed, and optiondly the probe may hybridize to the target.
  • This mixture of labeled probe and target is then passed to the second reaction chamber which contains the pool of probe-particles.
  • the free probe and probe-particles hybridize to target and appropriate probes are ligated in the second reaction chamber.
  • the ligating agent may be added at the reagent chamber or in either reaction chamber, preferably, the ligating agent is added in the second reaction chamber.
  • the probe-particle hybridization products in the second reaction chamber are andyzed as above.
  • the target nuddc acid is not amplified prior to andysis (either by PCR or in a vertor, e.g, a lambda library).
  • a vertor e.g, a lambda library
  • longer free and monkey probes are used in this embodiment because of the increase in sequence complexity of the sample (i.e., to distingdsh positives over background).
  • probe-particle embodiments described in this example are sdtable for use in any of the applications previously described, including, but not limited to the previously described diagnostic and sequencing applications. Additiondly, these probe particle embodiments may be modified by any the previously described variations or modifications. 67
  • the discrimination of perfect matches from mismatches in the binding of complementary polynucleotides is moddated by the addition of an agent or agents.
  • the complementary polynucleotides are a target polynudeotide and a polynucleotide probe.
  • the discrimination of perfect matches from mismatches may be modulated by adding an agent wherein the agent is a sdt such as tetraalkyl ammonium sdt (e.g., TMAC, Ricdli et al., Nucl.
  • a mixture of agents is added to the hybridization reaction to modulate the discrimination of perfect matches from mismatches. Some of these agents effect discrimination by reducing the entropy of mdting between two complementary strands.
  • the discrimination of perfe ⁇ matches from mismatches is improved by the agent or agents.
  • formamide a commody used denaturing agent
  • fo ⁇ namide a format III reaction was set up, and then varying amounts of fo ⁇ namide were added (0%, 10%, 20%, 30%, 40%, and 50%).
  • 0% a perfect match signd was detected and the background (mismatches) was high.
  • 10% formamide there was a good perfect match signd and the background/mismatch signd was reduced.
  • the perfert match signd was reduced (but detectable) and the background/mismatch signd was diminated.
  • 3O%-50% formamide there was no perfe ⁇ match or backgroundmismatch signd.
  • an agent is used to reduce or increase the T m of a pair of complementary polynudeotides.
  • a mixture of the agents is used 567 to reduce or increase the T w of a pair of complementary polynucleotides.
  • the agents may dter the T m in a number of ways, two examples, which are rtoi meant to limit the invention, are (1) agents which disrupt the hydrogen bonding between the bases of two complementary polynucleotides (Goodman, Proc. Nat'l Acad. Sci.94: 10493-10495 (1 97); Moran et al., Proc. Nat'l Acad. Sci.
  • an agent or agents are added to decrease the binding energy of GC base pairs, or increase the binding energy of AT base pdrs, or both.
  • the agent or agents axe added so that the binding energy from an AT base pdr is approximately equivdent to the binding energy of a GC base pair.
  • the energy of binding between two complementary polynudeotides is solely dependent on length.
  • the energy of binding of these complementary polynucleotides may be increased by adding an agent that neutrdizes or shields the negative charges of the phosphate groups in the polynudeotide backbone.
  • the discrimination of perfect matches from mismatches is enhanced in a format m reaction.
  • the discrimination is enhanced by an agent sele ⁇ ed from the group comprising a polyamine such as spe ⁇ mdine or spermine, other positively charged molecules which neutralize the negative chaige of the phosphate backbone, and a Mg " " " " ion.
  • the discrimination is dso enhanced by changing a physicd condition selected from the group comprising temperature, reaction time, or ionic strength, In a most prefe ⁇ ed embodiment, several agents are added and severd physicd conditions are changed. For example, the discrimination of perfe ⁇ matches from mismatches was increased about 10-100 fold by adding or dtering the following agents and 67
  • physicd conditions 100 mM MgCl 2 (increase from 10 M MgCl 2 ), 100 mM dithiothrietol, 100 ⁇ gml BSA (increase from 25 ⁇ g/ml), 10 mM ATT (increase from 1 mM), 10 mM spe ⁇ nadine, 10 umts/ ⁇ l ligase (increase from 4 units/ ⁇ l), at room temperature (increase from 4 °C) for 30 minutes (decrease from 120 minutes).
  • the MgCl 2 ATP, dithiothrietol, and BSA, a ⁇ to stabilize the ligase during the reaction.
  • the increased temperature and ligase concentration increase the rate at which ligation products are produced so that the reaction time can be decreased. These fartors may dso impact the ligation reaction through a kinetic effect.
  • the MgCl 2 , and the spe ⁇ nadine enhance discrimination by favoring the formation of perfe ⁇ matches over mismatches (they preferentially increase the ⁇ G of formation for the perfect match over the mismatch).
  • discrimination in a format III reaction was enhanced by the following agents: 20-100 mM MgCl 2 and 5- 10 mM ATP, 10-100 mM dithiothrietol, 50-100 ⁇ gtml BSA, or 5-20 mM spe ⁇ nadine.
  • Discrimination was dso enhanced by raising the temperature from 4 °C to 16 - 37 °C, or increasing the ligase concentration to 5-20 units/ ⁇ l.
  • the activity of a polynuclric acid polymerase is enhanced by the agents which enhance the discrimination of perfe ⁇ matches and mismatches between a target nucleic acid and a complementary polynucleotide.
  • the reaction mixture for the polymerase includes a targ ⁇ nudeic arid, a polynucleotide primer and an agent(s) which enhances the dscrimination of the perfect matches from the mismatches.
  • the polynucleic add polymerase reacts with the primer to replicate the target nucleic arid and co ⁇ ect (perfe ⁇ match) priming is favored over mismatch priming by the agent.
  • the agent(s) may include a sdt such as tetradkyl ammomum sdt (e.g., TMAC, Ricelli et al., Nucl.
  • polyamines such as spermidine and spermine (Thomas et al., Nucl. Adds Res.25:2396- 2402 (1997)), or other positively charged molecdes which neutralize the negative charge of the phosphate backbone, detergents such as sodium dodecyl sdfate, and sodium lauryl sarcosinate, minor/major groove binding agents, positivdy charged polypeptides, and intercalating agents such 567
  • acridine as acridine, ethidium bromide, and anthracine.
  • a mixture of agents is added to the hybridization reaction to moddate the discrimination of perfect matches from mismatches.
  • the agent(s) is used to enhance proper priming in a PCR reaction. For example, when 10 mM spe ⁇ nadine is added to a PCR reaction there was at least a 5-fold increase in product
  • the activity of polypeptide which modifies a nucleic acid is enhanced by the agents which enhance the dscrimination of perfe ⁇ matches and mismatches berween a target nucleic arid and a complementary polynucleotide.
  • the reaction mixture for the polypeptide includes a target nudeic acid, a complementary polynucleotide and an agent(s) which enhances the discrimination of the perfect matches from the mismatches.
  • the polypeptide reacts with the complex of the polynucleotide and the target nucldc acid and the perfect match complexes are favored over mismatch complexes by the agent Agents that maybe used to enhance dscrimirration include those recited supra.
  • the invention relates to modified DNA ligases which increase the discrimination of perfe ⁇ matches from mismatches for complementary polynucleotides.
  • the modified ligase enhances discrirnination in a number of ways, for example, the ligase may increase the difference in the oh rates and or the off rates between a perfert match product and a mismatch produ ⁇ (a kinetic effe ⁇ ); or the ligase may increase the binding energy difference between a perfe ⁇ match and a mismatch (a free energy [ ⁇ G] effert); or the ligase may itself discriminate between perfe ⁇ matches and mismatches ( ⁇ G or kinetic effect); or some combination of these and other factors.
  • the modified ligase of the invention may be prepared by methods well known in the art for modifiying polypeptides, such as are found in for e.g., Cu ⁇ ent Protocols in Protein Science (1997) JJ ⁇ Coligan, et al., eds. John Wiley & Sons, New York; and Kaiser ET, Lawrence DS, Rokita SE. (1985) "The chemicd modification of enzymatic specificity.” Annu Rev Biochem, 54:565-595. 67
  • the ligases of the invention may dso be modified by producing variants of the ligase nudeic acids.
  • These amino arid sequence variants may be prepared by methods known in the art by introduring appropriate nucleotide changes into a native or variant polynudeotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.
  • the amino arid sequence variants of the ligase nucleic acids are preferably constm ⁇ ed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature. These amino arid dterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).
  • Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino arid to a different hydrophobic ammo acid) and then with more distant choices (e.g., hydrophobic amino arid to a charged amino acid), and then deletions or insertions may be made at the target site.
  • conservative choices e.g., hydrophobic amino arid to a different hydrophobic ammo acid
  • more distant choices e.g., hydrophobic amino arid to a charged amino acid
  • Amino acid sequence ddetions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.
  • Amino arid insertions include amino- and/or carboxyl-termind fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino arid residues.
  • Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
  • termind insertions include the heterologous signd sequences necessary for secretion or for intracdlular targeting in different host cdls.
  • polynucleotides encoding the ligase nucleic acids are changed via site-dire ⁇ ed mutagenesis.
  • This method uses oligonucleotide sequences that encode the polynudeotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino arid to form a stable duplex on either side of the site of being dianged.
  • the tech ques of site-dire ⁇ ed mutagenesis are wdl known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al. , Dfcl ⁇ 2: 183 (1983).
  • a versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Rt>_;.10:6487-6500 (1982).
  • PCR may dso be used to create amino arid sequence variants of the ligase nudeic adds.
  • primer(s) that differs slightly 567
  • the co ⁇ esponding region in the template DNA in sequence from the co ⁇ esponding region in the template DNA can generate the desired a ⁇ no arid variant PCR amplification resdts in a population of product DNA fragments that differ from the polynucleotide template encoding the ligase at the position specified by the primer.
  • the produ ⁇ DNA fragments replace the co ⁇ esponding region in the plasmid and this gives the desired amino acid variant.
  • a further technique for generating amino acid variants is the cassette mutagenesis techdque described in Wdls et al , Qss 3.4.315 (19S5); and other mutagenesis techmques well known in the art, such as, for example, the techniques in Sambrook et al. , syjj ⁇ a, and Current Protocols jn Mplecular Biology. Ausubel etal.
  • the present invention further provides recombinant constructs comprising a modified ligase nucldc arid.
  • the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral ve ⁇ or, into whidi a modified ligase nucleic arid is inserted, in a forward or reverse orientation.
  • the ve ⁇ or may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
  • the ve ⁇ or may further comprise a marker sequence or heterologous ORF operably Jinked to an expression moddating fragment ("EMF") or uptake moddating fragment ("UMF").
  • sdtable vertors and promoters are known to those of skill in the art and are commercidly available for generating the recombinant constructs of the present invention.
  • the following ve ⁇ ors are provided by way of example.
  • Bacterid pBs, phagescript, PsiXl 74, pBluescript SK, pBs KS, pNH8a, pNH16a, pNHl 8a, pNH46a (Stratagene); pTrc99A pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • Promoter regions can be selected from any desired gene using CAT (chloramphericol transferase) ve ⁇ ors or other vectors with selectable markers.
  • Two appropriate ve ⁇ ors are pKK232- 8 and ⁇ CM7. Paiticdar named ba ⁇ erid promoters include lad, lacZ, T3, T7, gpt, lambda P R , and trc.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate ve ⁇ or and promoter is well within the levd of ordinary skill in the art 567
  • recombinant expression ve ⁇ ors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of £__ CQ I and S_ cerevisiae TRP 1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-fector, arid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracelldar medium.
  • the heterologous sequence can encode a fusion protein including an N-termind identification peptide imparting desired chara ⁇ eristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bartend use are constructed by inserting the modified ligase nucleic acid together with suitable translation initiation and te ⁇ nination signals in operable reading phase with a functiond promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure mdntenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation indude £_ colj.
  • useful expression ve ⁇ ors for ba ⁇ erid use can comprise a selectable marker and bacterid origin of replication derived from commercidly available plasmids comprising genetic dements of the well known cloning ve ⁇ or pBR322 (ATCC 37017).
  • commercid vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsda, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" se ⁇ ions are combined with an appropriate promoter and the structural sequence to be expressed.
  • the sdected promoter is derepressed by appropriate means (eg., temperature shift or chemicd induction) and cells are cultured for an additiond period.
  • appropriate means eg., temperature shift or chemicd induction
  • Cells are typicdly harvested by centrifugation, disrupted by physicd or chemicd means, and the resdting crude extra ⁇ retained for further purification.
  • Any host/vector system can be used to express the modified ligases of the present invention.
  • These indude but are not limited to, eukaryotic hosts such as HeLa cdls, Cv-l cell, COS cells, and Sf9 cdls, as well as prokaryotic host such as £_. c ⁇ ji and g. subtilfs-
  • the most preferred cdls are those which do not normally express the modified ligase or which expresses the modified ligase at a low natural level.
  • the modified ligase can be expressed in mammdian cdls, yeast, bacteria, or other cdls under the control of appropriate promoters.
  • Cell-free translation systems can dso be employed to produce the modified ligase using RNAs derived from the DNA constructs of the present invention.
  • Appropriate doning and expression vedors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al, in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, New York (1989), the disclosure of which is hereby incorporated by reference.
  • Various mammdian cell cdture systems can dso be employed to express the modified ligase.
  • Mammalian expression systems indude the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:115 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa andBHK cdl tines.
  • Mammdian expression vectors will comprise an origin of replication, a suitable promoter and , and dso any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, trans ⁇ iptiond termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic dements.
  • Recombinant modified ligase produced in bacterial cdture are usudly isolated by initial extraction from cell pdlets, followed by one or more sdting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liqmd chromatography (HPLC) can be employed for final purification steps.
  • Microbid cells employed in expression of roteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mecbanicd disruption, or use of cdl lysing agents.
  • the amino acid sequence can be synthesized using commercially avdlable peptide synthesizers. This is particdarly useful in producing smdl peptides 567
  • the modified ligase is purified from ba ⁇ erid cells which produce the modified ligase.
  • One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to purify the modified ligase of the present invention. These include, but are not limited to, immunochromatogiaphy, HPLC, size-exclusion chromatography, ion-exchange chromatography, and i ⁇ ununo-affinity chromatography.
  • the modified ligase of the present invention can dtematively be purified from cells which have been dtered to express the modified ligase.
  • a cell is sdd to be altered to express the modified ligase when the cell, through genetic manipdation, is made to product the modified ligase which it normally does not produce or which the cell normdly produces at a lower levd.
  • One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cdls in order to generate a cell which produces the modified ligase of the present invention.
  • the discrimination of perfect matches from mismatches is enhanced in a format III reaction.
  • the format III reaction the targ ⁇ nucleic arid interacts with complementary probes and the discrimination of perfe ⁇ matches from mismatches is enhanced by the modified ligase.
  • the modified ligase enhances discrimination in a number of ways, for example, the ligase may increase the difference in the on rates and/or the off rates between a perfert match product and a mismatch product (a kinetic effect - e.g., the ligase may preferentially bind to perfert matches and slow the off-rate of perfe ⁇ matches versus mismatches); or the ligase may increase the binding energy difference between a perfe ⁇ match and a mismatch (a free energy [ ⁇ G] effect - e.g., the ligase may preferentially bind to perfect matches and increase the stability of perfe ⁇ matches versus mismatches); or the ligase may itself discriminate between perfect matches and mismatches ( ⁇ G or kinetic effect - e.g., the modified ligase may ligate ody perfe ⁇ matches); or some combination of these and other fa ⁇ oxs. 567

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Abstract

L'invention a trait à des procédés utilisant une ADN ligase modifiée, qui permettent d'accroître une capacité de distinction entre des appariements parfaits et des mésappariements pour des polynucléotides complémentaires. La ligase modifiée permet d'améliorer la distinction de plusieurs façons, par exemple, en accroîssant et ou réduisant la différence de vitesse de réaction entre un produit d'appariement parfait et un produit de mésappariement (effet cinétique); en accroîssant la différence d'énergie de liaison entre un appariement parfait et un mésappariement (effet d'énergie libre [ΔG]); en établissant elle-même une distinction entre des appariements parfaits et des mésappariements (effet ΔG ou cinétique); ou en combinant ces propriétés et d'autres facteurs.
PCT/US1999/000176 1998-01-14 1999-01-14 Procede permettant d'ameliorer la mise en oeuvre d'une distinction entre des appariements parfaits et des mesappariements au moyen d'une adn ligase modifiee WO1999036567A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU25577/99A AU2557799A (en) 1998-01-14 1999-01-14 Enhanced discrimination of perfect matches from mismatches using a modified dna ligase

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US730098A 1998-01-14 1998-01-14
US09/007,300 1998-01-14

Publications (1)

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WO1999036567A2 true WO1999036567A2 (fr) 1999-07-22

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081743A2 (fr) * 2001-04-02 2002-10-17 Point-2-Point Genomics Ltd. Analyse de polynucleotides par pcr combinatoire

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081743A2 (fr) * 2001-04-02 2002-10-17 Point-2-Point Genomics Ltd. Analyse de polynucleotides par pcr combinatoire
WO2002081743A3 (fr) * 2001-04-02 2003-04-03 Brendan Hamill Analyse de polynucleotides par pcr combinatoire
US7771975B2 (en) 2001-04-02 2010-08-10 Point-2-Point Genomics Limited Polynucleotide analysis using combinatorial PCR

Also Published As

Publication number Publication date
AU2557799A (en) 1999-08-02

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