Van Ness, et al Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4504-9
Method for amplifying nucleic acid sequence.
Mukai,
et al US Patent Appl 20030073081 April 17, 2003
Virtually identical amplification techniques were conceived, and published to some extent at end of the last century. Particularly in mid 2000, in addition to filing Provisional Patent Applications, I personally presented the ideas described below to groups of scientists at three major West Coast biotechnology companies.
It is hoped that these amplifications, termed Nick Displacement Amplification (NDA), will continue to be developed, and will be freely available to anyone wishing to perfect and utilize them.
The following is a reproduction of a provisional
patent application I filed June 2001, which is
essentially a refiling of those filed in 2000. You
will need to scroll down through the first section
(Multiplex Analyses Utilizing Encoded Probes) which
itself is quite interesting.
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NUCLEIC ACIDS
1) Multiplex Analyses Utilizing Encoded Probes
2) Nick Displacement Amplification
3) Sequencing
4) Differentially Reactive Labels
This application comprises a refiling of of the following U.S. Provisional Applications by James Saba:
1) Ser. No. 60/210357 filed June 9, 2000, entitled "Isothermal Nucleic Acid Amplifications";
2) Ser. No. 60/211975 filed June 16, 2000, entitled "Nick Displacement Amplification (NDA)";
3) Ser. No. 60/218562 filed July 17, 2000, entitled "Nucleic Acids Detection and analysis";
4) Ser. No. 60/226565 filed August 21, 2000, entitled "Nucleic Acids Sequence Variation Analysis, and Sequencing"
The text of the first two, in their entirety, are present in the appendex of this application.
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Multiplex Analyses Utilizing Encoded Probes
Background of Invention
Analysis of a complex mixture is often facilitated by separating its
components via their distinct sizes or three dimensional structures.
Powerful separation techniques include chromatography, electrophoresis, mass
spectrometry, and polynucleotide hybridization.
Polynucleotides can be separated by both size (length or weight) and three dimensional structure (nucleotide sequence). Separation of polynucleotides is often integral to their sequencing.
Separation by size is utilized in commonly known sequencing techniques wherein multiple polynucleotide fragments are generated via nucleotide-specific chain termination or nucleotide-specific chemical cleavage. Separation by size can also be utilized in multiplex sequencing (identification) of polynucleotides of known sequence if there are prior known correlates between the sequences and their sizes.
Alternatively, separation by three dimensional structure can be utilized to sequence polynucleotides. Significant multiplex sequencing can be achieved by simultaneous hybridizations to microarrayed polynucleotides.
The ability to readily sequence polynucleotides, along with their variability and facile synthesis, makes them an attractive means for encoding probes (6172218, 6097103, 6060596, 6027889, 5985548, 5863722, 5770358, 5665539, 5635602, Fan et al, Gerry et al).
Shuber (5849483, 5834181, Shuber et al) teaches target sequence analyses utilizing an array of targets and oligonucleotide probes, comprising:
1) selectively amplifying a single gene in each of separate samples;Importantly, this process is carefully designed such that only one or rarely a few probes hybridize to, and are eluted from each arrayed target. This is because of the limited multiplexing capacity of the preferred and exemplified methods of sequencing eluted probes, which involve the electrophoretic size separation of polynucleotides resulting from chemical fragmentation or chain termination.
2) arraying the amplification products (targets) on a support;
3) presenting a mixture of allele-specific probes to the arrayed targets such that one or rarely a few of the probes hybridizes to each arrayed target; and
4) successively eluting and sequencing the allele-specific probe(s) which hybridized to each arrayed target.
As final sequencing alternatives he teaches "unique probe identifiers" and as a final example of these he teaches the following:
"One preferred form of this method is to collect the separated polymer, after hybridization to the immobilized nucleic acid target, into a tube containing oligonucleotides, each of which are complementary to one member of the polymer pool used to probe the target nucleic acid. In addition to a portion that is complementary to the polymer, the oligonucleotide also contains an additional sequence, the length of which is unique for that oligonucleotide."
Rephrased, he teaches hybridizing the eluted allele-specific polymer (primer) to one of a multitude of distinct oligonucleotides (extension templates). Critically, he does not teach multiplex hybridization of polymers (primers) to extension templates, nor identical oligonucleotides (extension templates) complementary to more than one member of the polymer (primer) pool.
In Shuber's probes, the sequences which prime the extension templates are intrinsic the target hybridizing sequences, and thus for each new target and probe a new extension template must be synthesized. Shuber does not envisage probes wherein "universal" encoded polynucleotide tags are conjugated to distinct target-directed ligands, nor the corresponding universally applicable extension templates. Likewise elusive to Shuber are ligands and/or targets which are not polynucleotides, and ligands directed to identical targets.
Shuber does not consider it necessary to carefully define the distinct oligonucleotide extension templates. For example, their length is not specified. Multiple dictionaries define an oligonucleotide as a "Linear sequence of up to 20 nucleotides joined by phosphodiester bonds." Distinctly, the extension templates of the present invention are of essence, and they are preferably identical and much longer. It will be made abundantly clear that the multiplexing capacity of the present invention, in large measure resultant from utilizing novel extension templates, vastly exceeds any of the methods of Shuber.
Finally and importantly, Shuber does not teach primer derivation via probe cleavage or limited polymerase extension. Nor separation of primer extension products by weight.
Consistent with Shuber's above quoted cursory description, the contents thereof is not claimed, nor included in the refined and detailed journal article.
There remains a need for simple, versatile and extensive multiplexing processes; clearly delineated so as to direct future scientific endeavors and discoveries.
Brief Summary of Invention
Novel combinations of routinely utilized technology, providing versatile and
practical methods and kits for extensive multiplex sequencing of encoded
polynucleotides, heretofore not envisaged without the use of microarrays. Of
essence are encoded primers and uniquely designed polynucleotide extension
templates. Applicable to both qualitative and quantitative analyses, the
methods are particularly useful in multiplex analyses of nucleic acid
sequence variations such as single nucleotide polymorphisms (SNP), multiplex
qualitative and quantitative analyses of mRNA expression, and in the tagging
of complex library members.
Brief Description of Drawings
Figures 1A & 1B. Encoded primer derivation by limited polymerase extension of probes.
Figure 2. Encoded primer derivation by probe ligation.
Figure 3. Encoded primer derivation by probe cleavage.
Detailed Description of the Invention
Disclosed herein are methods and kits for simultaneously preforming multiple
analyses wherein each of the following process steps is done in multiplex:
i) presenting a mixture of distinct encoded probes to targets under conditions conducive of specific probe-target interactions;"Multiplex" is descriptive of a process wherein multiple distinct reactions or separations are conducted simultaneously within a single containment.
ii) deriving distinct encoded primers from those distinct probes which specifically interacted with targets;
iii) hybridizing derived distinct encoded primers to extension templates;
iv) extending hybridized distinct encoded primers to form extension products of predetermined and distinct size;
v) separating distinct-sized extension products, or products thereof, by size.
"Distinct" is descriptive of an entity or process which in some intrinsic characteristic is different from others. Two identical entities or processes which are separated are not distinct. An unqualified statement such as "targets" or "extension templates" optionally indicates multiple identical or distinct entities. For clarity, when describing a process involving a mixture of distinct reagents or products, for example "distinct encoded primers", only one of each distinct reagent or product is being referred to. In practice however, each distinct reagent or product occurs in multiple.
"Polynucleotide" refers to a linear polymer of any length, which may reside in a larger polymer, and which can hybridize with a complementary polymer such that the monomers at each position along the resultant hybrid are matched. Preferably polynucleotides are single stranded DNA, RNA, and/or variants thereof. A polynucleotide, such as peptide nucleic acid (PNA), need not be charged. Polynucleotide size is defined by "length"(number of composite nucleotides) or "weight" (number and molecular weights of composite nucleotides).
"Nucleotide" denotes a monomer which resides in or has the potential to reside in a polynucleotide.
Henceforth "probe" or "encoded probe" comprises a polynucleotide code. In certain polynucleotide probes this code is intrinsic the target-directed portion of the probe. Alternatively the code is intrinsic a polynucleotide "tag" which is conjugated to a target-directed "ligand". Conjugation of a tag to its ligand may or may not be covalent, and optionally involves a cleavable linkage. Conjugation may be mediated by a support, such as a bead, to which multiple copies of both tag and ligand are affixed. It may be preferable to have the tags in duplex, perhaps hairpin form, for example to keep them rigid or masked during contacting probes with targets. Since tags are not decisive in target-probe interactions, they are "universally" applicable to distinct sets of ligands, and allow for universally applicable extension templates.
Both targets and ligands can be vastly diverse in constitution, and most obviously comprise polynucleotides, peptides, polypeptides, carbohydrates, lipids, and/or relatively small synthetic organic molecules.
Probes may be modified, for example to impart nuclease resistant, enhance hybridization, allow for amplification and/or purification of primers or extension products.
Targets and probes may be covalently or noncovalently conjugated to a support, optionally involving a cleavable linkage. Numerous supports are well know and can be of various configurations, composed of various materials, and include soluble polyvalent polymers. A target particularly may be part of a relatively large assembly such as a cell's chromosome or surface.
Henceforth "primer" or "encoded primer" comprises a polynucleotide code which directs the primer's hybridization to an extension template. Preferably, distinct primers hybridize to extension templates with high specificity. Variations in hybrid thermal stability can be minimized by utilizing modified nucleosides and chaotropic agents (Nguyen et al/1999). Multiple identical primers can be derived from one target (6121001, 6001567, 5882867, 5869252, 5660988, Okano et al).
"Primer extension template" or "extension template" denotes a linear or circular polynucleotide which is distinct from targets or variants thereof, and guides extension of a primer to a predetermined size. Primer extension to a definite size can result from run-off polymerization or by obstructing polymerase progression. Preferably, when utilizing run-off polymerization and electrophoretic separation of extension products to single base resolution, the extension template is linear and about 1000 nucleotides or less. The sequence of the extension template should not be such as to result in intra- or intermolecular structures which impede polymerase progression, cause premature cleavage or template switching, nor inhibit reannealing and elongation of a displaced incompletely extended primer (Nguyen et al/2000, Tombline et al, Guieysse et al, Patel et al, Clark). Repetitive sequences particularly can be problematic. The arrangement of distinct primer binding sequences along the template can vary from being totally separate to extensively overlapping (Caskey et al).
Extension templates can, as probes, be modified in several ways. One such modification would be to have 3' termini which cannot be extended. Another example, it can be modified so as to allow removal from extension products prior to their separation. Yet another example would be the presence of universal bases within a primer-binding sequence such that distinct primers can utilize this sequence.
Preferably distinct primers prime identical extension templates provided in excess. One means by which distinct primers can prime identical extension templates to result in distinct-lengthed products is when the primers have distinct length and prime the extension templates identically. Alternatively, distinct primers utilize distinct primer-binding sites (see Figure 1A).
MedLine and patent database searches uncovered numerous computer programs and schemes for selection of optimal hybridizing sequences (e.g., 6143497, 6138077, 6083695, Mei et al, Mitsuhashi, Warnon et al) and these are valuable in designing probes and extension templates.
Primer extension preferably is achieved by polymerase incorporation of nucleoside triphosphates or variants thereof. Alternative means of primer extension include nucleotide self assembly or polynucleotide ligation.
Polymerases with utility in primer extension include, but are not limited to, Vent (exo-), Deep Vent (exo-), Bst (large fragment), Bca (exo-), phi 29 and T7 (exo-). Combinations of polymerases (Barnes, Wu et al) as well as techniques of hot-start polymerization (6183998) may be advantageous.
Nucleotide variants such as deoxyinosine and 7-deazaguanosine triphosphates, the presence or concentration of cations, and diverse reagents such as betaine, DMSO, gelatin, lipids, Perfect Match® (Stratagene), processivity factors, single-stranded binding protein (SSB) and RecA may aid in synthesizing the desired complete, precisely initiated and terminated extension products.
"Primer extension product" or "extension product" indicates the primary extension product. Unless discernible labels are utilized, distinct encoded primers are extended to form extension products of predetermined and distinct size. Rephrased, for a distinct primer, extension of a multitude of primers thereof results in extension products distinguishable from the extension products of other distinct primers. Preferably the extension products of identical primers are identical in length (6090590, Livak et al).
Primary extension products can be subjected to amplification, for example, by designing the primer and/or its extension template to have or encode for an RNA polymerase promoter.
Preferably extension products are distinct in length and electrophoretically separated. Alternatively distinct weights can be the basis of separation. When separating by weight, certain modifications to the extension products can facilitate separation. For example, identically lengthed extension products can be separated electrophoretically if they contain distinct numbers of mobility altering nucleotide derivatives. Similarity, weight altering modifications can facilitate separation via mass spectrometry.
"Labels" facilitate detection and allow discernment of distinct yet unseparated extension products. Labels are are preferably fluorescent dyes covalently conjugated to primers or nucleotide substrates. Particularly promising are mass labels (Xu et al).
To exemplify the exceptional multiplexing capacity of the present invention, if extension products are separated electrophoretically in a capillary tube to resolve 500 distinct-lengthed polynucleotides, utilizing 5 distinguishable fluorescent dyes would allow 2500 (500 x 5) encoded primers to be sequenced. Considering that current DNA sequencing machines use close to 100 such capillary tubes, an astonishing quarter million (250,000) analyses could be simultaneously processed.
Unlike microarrays, the present invention has considerable quantitative capacity, particularly as it relates to mRNA expression analyses.
Turning to the figures, note the sequences of probes, targets, primers and extension templates involved in hybridization are exaggerated as elongated rectangles.
In Figures 1A and 1B limited primer extension (minisequencing) results in formation one or more exonuclease resistant internucleoside linkages (e.g., phosphorothioate, phosphoramidate, or boranophosphate) at the 3' ends of the probes (Syvanen). Only those probes extended resist subsequent 3' exonuclease digestion and go on to prime the extension templates. Run-off polymerization extends the hybridized primers to predetermined and distinct lengths. Extension products are subsequently separated via electrophoresis under denaturing conditions.
In Figure 1B, the incorporated nucleotide is also labeled. For clarity, in this and subsequent figures only one successful primer derivation is delineated.
Figure 2 exemplifies deriving a primer by ligation (6027889). After successful target hybridization and enzymatic or chemical ligation, the 5' exonuclease resistant modification (cap) prevents the digestion of the universal 3' tag sequence, which subsequently primes the extension template. Labeled nucleotides are incorporated during primer extension.
In the above examples the exonuclease also conveniently digests the target nucleic acids. Alternatively or in addition to modifications imparting exonuclease resistance, modifications enabling primer isolation can be utilized.
Figure 3 exemplifies deriving an encoded primer by invasive or invader-directed probe cleavage (6001567). When the two probes are appropriately hybridized to target, the tagged probe is cleaved to liberate the tag, which subsequently primes the extension template. At elevated temperatures there is rapid turnover of probes, resulting in multiple identical primers per target molecule. Optionally uncleaved tagged probes can be removed, such as by their 3' modification (e.g., biotin).
Cycling probe technology (5660988, Warnon et al) is another means of deriving multiple identical primers per target via probe cleavage.
Further examples of primer derivation would involve cleavage of internally or terminally mismatched probes (Bazer et al), and pyrophosphorolysis of 3' terminal nonextendable nucleotides (Liu et al).
Provisional Claims
1.1) An analysis utilizing a mixture of distinct encoded probes, comprising the following multiplex processes:Referencesi) presenting the mixture of distinct encoded probes to targets under conditions conducive of specific probe-target interactions;1.2) The analysis of (1) wherein the extension templates are identical.
ii) deriving distinct encoded primers from those distinct probes which specifically interacted with targets;
iii) hybridizing derived distinct encoded primers to extension templates;
iv) extending hybridized distinct encoded primers to form extension products of predetermined and distinct length;
v) separating distinct-lengthed extension products, or products thereof, by size.
1.3) The analysis of (1) wherein more than a few distinct primers are derived and extended.
1.4) The analysis of (1) wherein the extension templates are greater than 100 nucleotides in length.
1.5) The analysis of (1) wherein primer derivation involves phosphodiester bond cleavage.
1.6) The analysis of (1) wherein primer derivation involves limited polymerase extension of probes.
1.7) The analysis of (1) wherein the primary primer extension products are amplified.2.1) An analysis utilizing a mixture of distinct encoded probes, each comprising a target-directed ligand conjugated to an encoded polynucleotide tag, comprising the following multiplex processes:
i) presenting the mixture of distinct encoded probes to targets under conditions conducive of specific ligand-target interactions;
ii) deriving distinct encoded primers from those distinct probes whose ligands specifically interacted with targets;
iii) hybridizing derived distinct encoded primers to extension templates;
iv) extending hybridized distinct encoded primers to form extension products of predetermined and distinct length;
v) separating distinct-length extension products, or products thereof, by size.2.2) The analysis of (2.1) wherein the extension templates are identical.
2.3) The analysis of (2.1) wherein more than a few distinct primers are derived and extended.
2.4) The analysis of (2.1) wherein the extension templates are greater than 100 nucleotides in length.
2.5) The analysis of (2.1) wherein primer derivation involves phosphodiester bond cleavage.
2.6) The analysis of (2.1) wherein primer derivation involves phosphodiester bond formation.
2.7) The analysis of (2.1) wherein the primary primer extension products are amplified.
2.8) The analysis of (2.1) wherein the ligands are not polynucleotides.
2.9) The analysis of (2.1) wherein the targets are not polynucleotides.
2.10) The analysis of (2.1) wherein the targets are identical.3.1) An analysis utilizing a mixture of distinct encoded probes, comprising the following multiplex processes:
i) presenting the mixture of distinct encoded probes to targets under conditions conducive of specific probe-target interactions;3.2) The analysis of (3.1) wherein each distinct encoded probe comprises a distinct target-directed ligand conjugated to a distinct encoded polynucleotide tag.
ii) deriving distinct encoded primers from those distinct probes which specifically interacted with targets;
iii) hybridizing derived distinct encoded primers to extension templates;
iv) extending hybridized distinct encoded primers to form extension products of predetermined and distinct weight;
v) separating distinct-weightd extension products, or products thereof, by weight.
3.3) The analysis of (3.1) wherein the distinct-weighted extension products have identical length.4.1) A kit useful in performing a prior claimed analysis comprising:
i) a mixture of distinct encoded probes; and5.1) A kit useful in performing a prior claimed analysis comprising: i) a mixture of distinct encoded probes, each comprising target-directed ligand conjugated to an encoded polynucleotide tag; and ii) linear extension templates.
ii) identical linear extension templates.
5.2) The kit of (5.1) wherein the linear extension templates are identical.
The following publications are incorporated by reference. They more fully describe the state of the art to which this invention pertains, and teach applicable material and methods.
6183998 Feb 6, 2001 Ivanov et al 435/91.2. "Method for reversible modification of thermostable enzymes"
6172218 Jan 9, 2001 Brenner 536/25.4. "Oligonucleotide tags for sorting and identification"
6153379 Nov 28, 2000 Caskey et al 435/6. "Parallel primer extension approach to nucleic acid sequence analysis"
6150516 Nov 21, 2000 Brenner et al 536/24.3. Kits for sorting and identifying polynucleotides
6143497 Nov 7, 2000 Dower et al 435/6. "Method of synthesizing diverse collections of oligomers"
6138077 Oct 24, 2000 Brenner et al 702/19. "Method, apparatus and computer program product for determining a set of non-hybridizing oligonucleotides"
6121001 Sept 19, 2000 Western et al 435/6. "Detection of nucleic acids by target-catalyzed product formation"
6090590 July 18, 2000 Kao 435/91.1. "Reducing nontemplated 3' nucleotide addition to polynucleotide transcripts"
6087103 July 11, 2000 Burmer 435/6. "Tagged ligand arrays for identifying target-ligand interactions"
6083695 July 4, 2000 Hardin et al 435/6 "Optimized primer library for gene sequencing and method of using same"
6060596 May 9, 2000 Lerner et al 536/25.3. "Encoded combinatorial chemical libraries"
6027889 Feb 22, 2000 Barany et al 453/6. "Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions"
6001567 Dec 14, 1999 Brow, et al 435/6. "Detection of nucleic acid sequences by invader-directed cleavage"
5985548 Nov 16, 1999 Collier et al 435/6. "Amplification of assay reporters by nucleic acid replication"
5882867 Mar 16, 1999 Ullman et al 435/6. "Detection of nucleic acids by formation of template-dependent product"
5869252 Feb 9, 1999 Bouma et al 435/6. "Method of multiplex ligase chain reaction"
5863722 Jan 26, 1999 Brenner 435/6. "Method of sorting polynucleotides"
5849483 Dec 15, 1998 Shuber 435/5. "High throughput screening method for sequences or genetic alterations in nucleic acids"
5834181 Nov 10, 1998 Shuber 435/5. "High throughput screening method for sequences or genetic alterations in nucleic acids"
5770358 June 23, 1998 Dower et al 435/6. "Tagged synthetic oligomer libraries"
5665539 Sept 9, 1997 Sano et al 435/6. "Immuno-polymerase chain reaction system for antigen detection"
5660988 Aug 26, 1997 Duck et al 435/6. "Cycling probe cleavage detection of nucleic acid sequences"
5635602 June 3, 1997 Cantor et al 530/391.1. "Design and synthesis of bispecific DNA-antibody conjugates"
Barnes "PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates" Proc Natl Acad Sci U S A 1994 Mar 15;91(6):2216-20
Bazer et al "Mutation identification DNA analysis system (MIDAS) for detection of known mutations" Electrophoresis 1999 Jun;20(6):1141-8
Fan et al "Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays." Genome Res 2000 Jun;10(6):853-60
Fei et al "Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry" Rapid Commun Mass Spectrom 2000;14(11):950-9
Gerry et al, "Universal DNA microarray method for multiplex detection of low abundance point mutations." J Mol Biol 1999 Sep 17;292(2):251-62
Guieysse et al "Oligonucleotide-directed switching of DNA polymerases to a dead-end track" Biochemistry 1995 Jul 18;34(28):9193-9
Liu et al "Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification"Biotechniques 2000 Nov;29(5):1072-6, 1078, 1080
Livak et al "Detection of single base differences using biotinylated nucleotides with very long linker arms" Nucleic Acids Res 1992 Sep 25;20(18):4831-7
Mei et al "Octamer-primed sequencing technology: development of primer identification software" Nucleic Acids Res 2000 Apr 1;28(7):E22
Mir et al "Sequence Variation in Genes and Genomic DNA: Methods for Large-Scale Analysis" Annu Rev Genomics Hum Genet. 2000. 1:329-360
Mitsuhashi "Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences" J Clin Lab Anal 1996;10(5):277-84
Nguyen et al "Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents" Nucleic Acids Res 1999 Mar 15;27(6):1492-8
Nguyen et al "Minimizing the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation." Nucleic Acids Res 2000 Oct 15;28(20):3904-9
Okano et al "DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA" Anal Biochem 1995 Jun 10;228(1):101-8
Pastinen et al "Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation" Clin Chem 1996 Sep;42(9):1391-7
Patel et al "Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide" Proc Natl Acad Sci U S A 1996 93(7):2969-74
Clark "DNA synthesis on discontinuous templates by DNA polymerase I of Escherichia coli" Gene 1991 Jul 31;104(1):75-80
Shuber et al "High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes" Hum Mol Genet 1997 Mar;6(3):337-47
Skerra "Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity." Nucleic Acids Res 1992 Jul 25;20(14):3551-4
Syvanen "From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms" Hum Mutat 1999;13(1):1-10
Tombline et al "Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops." Proc Natl Acad Sci U S A 1996 Apr 2;93(7):2724-8
Warnon et al "Colorimetric detection of the tumberculosis complex using cycling probe technology and hybridization in microplates" BioTechniques 2000 June 28(6):1152-07 (See references 5, 10 & 14 therein)
Wu et al "Shadow bands in PCR amplification of trinucleotide repeats and their elimination" [Chinese] Chung Hua I Hsueh I Chuan Hsueh Tsa Chih 1998 Feb 10;15(1):42-5
Xu et al "Electrophore mass tag dideoxy DNA sequencing" Anal Chem 1997 Sep 1;69(17):3595-602
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Nick Displacement Amplification (NDA)
Background of Invention
Nucleic amplification technology has been essential to the advancement of
molecular biology. Initially bacterial plasmids and viruses where harnessed
to amplify select DNA fragments. Subsequent skillful purification and use of
RNA and DNA polymerases allowed efficient amplifications in vitro. The PCR
is an important versatile means of in vitro amplification yet its dependence
on thermal cycling apparatus encumber its application. Furthermore, PCR is
seriously limited in its multiplexing capacity. So being there have been
several attempts to devise amplification techniques which are isothermal and
capable of simultaneously amplifying a large number of multiple sequences
simultaneously.
Richards et al (5650271) reports isothermal enzymatic synthesis of oligonucleotides using a modified synthetic template to which a primer is hybridized to and extended along. The primer extension product of the resultant duplex is nicked at the hemimodified site, followed by the thermal dislodging of the 3' portion. Subsequently, the 5' portion is again extended to create a new primer extension product and a rejuvenated nicking site. Note thermal denaturation significantly limited the length of the amplification product.
Particularly relevant to the present invention are those isothermal amplifications which rely on strand displacement by the polymerase rather than thermal denaturation as the means of separating duplex DNA.
One of the simplest and most efficient means of isothermal strand displacement amplification is by rolling circle amplification. An enhanced derivative thereof, utilizing two primers, was delineated in 1991 (Saba).
Fraiser et al (5648211), Walker et al (5270184, 5712124) and Wick et al (6063604) describe strand displacement amplifications which require: 1) 5' tailed primers, 2) modified nucleotides to produce hemimodified duplexes, and 3) restriction enzymes to selectively nick the nonmodified strand.
Cleuziat et al (5824517) teach a strand displacement amplification wherein an RNA primer is extended with deoxynucleotides, and then hydrolyzed from the primer extension product by RNase H. Subsequently a new RNA primer hybridize in its place, and its extension results in the displacement of the previously synthesized strand.
Kainz et al teach a version of nick translation for detecting a specific mRNA which involves presenting a mixture of mRNA to an immobilized ssDNA under hybridization conditions. If the specific mRNA of interest is present and hybridized to the ssDNA, it is nicked nonspecifically with RNase H, and then the resultant 3' termini are extended with labeled nucleotides via DNA polymerase I.
Kacian et al (5916777) teach synthesis of oligonucleotides utilizing a reusable ssDNA primer which has a 3' modification allowing selective cleavage from the primer extension product. In a preferred embodiment a ssDNA primer is modified by inclusion of a 3' terminal ribonucleotide, and the primer extension product is cleaved by alkali or an RNase. Concurrently with cleaving the primer extension product to provide the desired oligonucleotide and reusable primer, preferably the template is also hydrolyzed to facilitate purifications. The recovered primer is then again contacted with an extension template and the process is repeated.
Finally, Bishop et al describe a plasmid cloning vector wherein one of the strands is selectively nicked by certain preparations of EcoRI, and the utility thereof in strand separation.
There remains a need for simple efficient nucleic acid amplifications, particularly isothermal amplifications.
Brief Summary of Invention
Versatile and specific isothermal nucleic acids amplifications which do not
require expensive modified nucleotides, modified templates, or a tailed
primers. Preferably primers have modifications which direct nicking and the
endonucleases utilized rarely if ever cleave nontarget sequences.
Conjointly, dsDNA cloning vectors comprising a nicking site(s), and kits
thereof, are disclosed.
Brief Description of Drawings
Figures 4A & 4B. NDA wherein the target is ssDNA or dsDNA with definite downstream termini.
Figure 5. NDA to amplify a dsDNA section residing in a dsDNA with indefinite termini.
Figure 6. NDA utilizing modified primers to amplify a dsDNA section residing in a dsDNA with indefinite termini.
Figure 7. NDA to amplify a ssDNA section within a dsDNA with indefinite ends.
Figure 8. NDA utilizing an RNA target.
Figure 9. NDA utilizing a ssDNA circular target.
Detailed Description of the Invention
Disclosed herein is "Nick Displacement Amplification" (NDA) is a highly
versatile and specific means of strand displacement amplification which
preferentially utilizes uniquely designed primers, unmodified targets, and
nonmodified nucleotides. Being of similar nature to the strand displacement
amplifications as taught by Fraiser et al (5648211), Walker et al (5270184,
5712124) and Wick et al (6063604) many of the materials and processes
utilized in performing NDA have fortuitously been described.
NDA is primarily a process for synthesizing a polynucleotide with
complementarity to a duplexed target polynucleotide, devoid of a
modification which appreciably influences nicking, comprising:
i) contacting the duplexed target with a nicking agent such that the nontarget strand is selectively nicked at a prescribed location;The "target polynucleotide" is a linear or circular single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) which does not contain a modification which appreciably influences nicking. Duplexed targets include those which occur in freshly isolated nucleic acids and those which result from in vitro polymerization. In vivo NDA utilizing targets residing in living cells is conceivable.
ii) extending the 3'-ended fragment of the nick with a polymerase such that the nicking site is rejuvenated and the 5'-ended strand of nick is displaced; and
iii) repeating steps (i) and (ii) such that there are are multiple cycles of nicking, extension and displacement.
In a preferred embodiment the duplexed targets originate from the priming of a target with a modified primer. One type of primer modification is a 5' tail which when made duplex is a substrate for a rare nicking endonuclease. Another type of primer modification occurs within the sequence which hybridizes to the target, and which directs nicking within or adjacent to the primer. These modifications can be nucleotide variants or mismatched nucleotides, recognized by mutant restriction enzymes or repair endonucleases. Alternatively a ssDNA primer can be modified by substituting one or more deoxyribonucleotides with ribonucleotides, the nicking agent therewith preferably being an RNase such as RNase H.
Precise nicking within a primer may be facilitated by making certain of its internucleoside bonds resistant to hydrolysis, such as by converting them to phosphorothioate, boranophosphate, methylphosphonate, or peptide bonds. For example excision repair nucleases generally nick both 5' and 3' the lesion, and the 5' cleavage could be prohibited. Further, some RNase Hs do not exclusively cleave at the RNA-DNA junction, and other susceptible phosphodiester bonds can be made resistant.
Primer modifications which do not direct nicking, such as labels, separation appendages, obstructions to polymerase progression, exonuclease resistant or nonextendable termini, internucleoside bond variants, and hybridization enhancers or stabilizers also have utility.
Primers or targets may be covalently or noncovalently conjugated to a support, perhaps via a cleavable linker. Numerous supports are well know and can be of various configurations, composed of various materials, and include soluble polyvalent polymers.
Nicking agents are preferably molecules, yet conceivably physical forces such as light could effect nicking. While molecular nicking agents include a large variety of substances, including relatively small molecules and ribozymes, they are preferably proteasous endonucleases such as restriction enzymes, ribonucleases, homing endonucleases, and various other endonucleases involved in DNA repair, replication, recombination and/or topology. A nicking agent can be conjugated to a target-directing molecule such as a transcription factor.
Nicking agents with utility in NDA selectively nick the nontarget strand at least 100 times more efficiently than the target strand. Preferably nicking occurs exclusively in the nontarget strand.
Conveniently, selective nicking agents such as the thermophilic N.BstNB I (New England Biolabs®) are now becoming commercially available. Preferably however the nicking agent's recognition sequence is rarer than that of N.BstNB I.
Alternatively, several rare nicking endonucleases have utility. These include retrotransposon endonucleases such as insect R2 and Xenopus Tx1L (Christensen et al); mutants of homing enzyme PI-Sce I (Christ et al); and a mitochondrial replication origin nicking enzyme (Parks et al).
Certain RNase H endonucleases such as those from yeast (Karwan et al) and avian myeloblastosis virus (Champoux et al) preferentially nick at the RNA-DNA junction of an RNA-DNA/DNA hybrid. Certain other RNase Hs nonpreferably cleave at the RNA-DNA junction, yet with modification of other ribonucleotide phosphodiester bonds, if present, cleavage can be restricted to the DNA-RNA junction. Noteworthy, affixing RNase H to oligonucleotides directs nicking (Bekkaoui et al). Unlike RNase Hs, other RNases leave a 3' phosphate, and addition of a second enzyme such as T4 polynucleotide kinase to remove this 3' phosphate can allow for the use of these RNases in NDA.
Such ribonucleotide modified ssDNA primers, being independent of a nicking agent recognition sequence, are exceptionally versatile. This is also the case for primers which contain a modification or lesion such as deoxyinosine or pyrimidine dimers which are recognized by repair endonucleases.
Certain mutant and wild-type restriction enzymes preferentially nick the modified strand of a hemimodified duplex, rather than the commonly encountered reverse scenario (Lanio et al, Sheflian et al, Voigt et al). For like restriction enzymes to be useful in NDA, they preferably are mutants which are selective to the modified strand, and they need nick 3' (downstream) the modification such that the nicking site is rejuvenated.
Potentially applicable DNA polymerases include Vent (exo-), Deep Vent (exo-), Pfu (exo-), Bst (large fragment), Bca (exo-), phi 29, T7 (exo-), and Klenow (exo-). Thermophilic polymerases and/or highly processive polymerases are likely advantageous.
Types and concentrations of monovalent and divalent cations, and diverse reagents such as betaine, DMSO, gelatin, lipids, Perfect Match® (Stratagene) processivity factors, single-stranded binding protein (SSB), and RecA, may facilitate strand displacement.
The polynucleotide products of NDA are complementary to at least a portion of the target polynucleotide, and can subsequently function as probes, primers, or targets. "Primer extension product" indicates the unnicked primer extension product, while "extension product" indicates only the 3' portion of the nicked primer extension product.
Polymerization of variant nucleotides has utility. For example preforming NDA in the presence of chain terminating nucleotides is an attractive alternative to thermal cycle sequencing. Furthermore, incorporation of variant nucleotides such as 5'-alpha-thiotriphosphates, hydroxymethyl dUTP and deoxyinosine triphosphate destabilizes the extension product and may facilitate entry of the polymerase at the nick. Destabilizing the extension product could also be facilitated by selecting target sequences such that the 5' end of the extension product is rich in adenine and thymine.
Turning to the figures, note the sequences involved in hybridization are exaggerated as elongated rectangles. The nucleic acid representations within these figures do not necessarily describe the exact nature of reaction intermediates nor the exact chronological order of events.
Figure #4 exemplifies NDA using either a primed ssDNA target or an intact dsDNA target, both with definite downstream ends. Note the primer does not have a 5' tail, targets are not modified, and modified nucleotides are not utilized. For the ssDNA target, primer hybridization results in appearance of a nicking site within the unmodified primer. Note that this primer is "catalytic" and gives rise to multiple ssDNA extension products.
Figure #5 exemplifies the amplification of a dsDNA insert within a dsDNA with indefinite ends, perhaps a plasmid cloning vector. Initially both nicking sites are nicked, and with simultaneous extension the insert is duplicated. The ssDNA NDA products resultant from each of these inserts hybridize to form a multitude of the dsDNA insert. Further amplification could be achieved with the inclusion of nickable primers which target the NDA ssDNA amplification products.
Alternatively to amplifying a dsDNA section, a strand-specific ssDNA section could be amplified if the nicking sites are responsive to distinct nicking agents, and an appropriate restriction enzyme is used to cleave at one end of the section (see Figure #1B).
Figure #6 exemplifies utilizing terminally modified primers in the amplification of a dsDNA segment residing in a dsDNA with indefinite ends. For clarity only one of the displacement products from the first amplifications is shown. Further amplification, resulting in heterogeneous products could be achieved if nicking occurred sufficiently internal the primer such that the final ssDNA extension products are themselves primed. Although similar to PCR, NDA is patentably distinct in that it results in extension products rather than primer extension products.
Figure #7 exemplifies the use of NDA to produce ssDNA sections from a dsDNA target with indefinite ends. A further level of amplification utilizing a second nickable primer is optional.
In Figure #8 ssRNA is the target and one tailed and one nonnickable primer are combined to produce a dsDNA amplification product. Alternatively a ssDNA amplification product can result if the second nonnickable primer had a modification preventing it from acting as a template.
Figure #9 exemplifies NDA utilizing a circular target. Therein, this closed circular ssDNA target is hybridized with a nickable primer, which is subsequently extended with a highly processive strand-displacing polymerase. As extension is occurring the primer is nicked and another polymerase engages. This nicking and polymerase engagement continue until the template is loaded with polymerases. A similar amplification occurs with strand-specific NDA of a circular dsDNA target.
Note also that with a circular dsDNA target, nicking one or the other strand in the presence of exonuclease rather than polymerase, would exclusively provide the nonnicked circular ssDNA strand.
Provisional Claims
1) A process of synthesizing a polynucleotide with complementarity to a duplexed target polynucleotide devoid of modification which appreciably influences nicking, comprising:Referencesi) contacting said duplexed target with a nicking agent such that the nontarget strand is selectively nicked at a prescribed location;2) The process of (1) wherein the nontarget strand has a modification which directs nicking.
ii) extending the 3'-ended fragment of the nick with a polymerase such that the nicking site is rejuvenated and the 5'-ended strand of nick is displaced; and
iii) repeating steps (i) and (ii) such that there are are multiple cycles of nicking, extension and displacement.
3) The process of (2) wherein the nicking agent is a mutant restriction enzyme.
4) The process of (2) wherein the nicking agent is not a restriction enzyme.
5) The process of (4) wherein the nicking agent is a repair endonuclease, or mutant thereof.
6) The process of (4) wherein the nicking agent is an RNase, and the nontarget strand is ssDNA modified by substituting one or more deoxyribonucleotides with ribonucleotides.
7) The process of (1) wherein the nicking agent is not a restriction enzyme.
8) The process of (7) wherein the nicking agent is a homing endonuclease, or mutant thereof.2.1) A process for synthesizing a polynucleotide with complementarity to a target polynucleotide comprising:
i) hybridizing said target with a primer which has a modification which directs nicking;2.2) The process of (2.1) wherein the nicking agent is a mutant restriction enzyme.
ii) nicking within or adjacent to hybridized primer;
iii) extending the 3'-ended fragment of the nick with a polymerase such that the nicking site is rejuvenated and the 5'-ended strand of nick is displaced; and
iv) repeating steps (iii) and (iv) such that there are multiple cycles of nicking, extension and displacement.
2.3) The process of (2.2) wherein the nicking agent is not a restriction enzyme.
2.4) The process of (2.3) wherein the nicking agent is a repair endonuclease, or mutant thereof.
2.5) The process of (2.3) wherein the nicking agent is an RNase, and the nontarget strand is ssDNA modified by substituting one or more deoxyribonucleotides with ribonucleotides.3.1) The process of (1) or (2.1) wherein the polymerized nucleotides comprise chain terminating nucleotides.
4.1) The process of (1) or (2.1) wherein the target is a circular ssDNA.
5.1) A process of deriving a specific strand of a covalently closed circular dsDNA cloning vector, comprising:
i) selectively nicking one strand; and5.2) A kit comprising:
ii) digesting the nicked strand with an exonuclease.1) a dsDNA cloning vector;6.1) A dsDNA cloning vector wherein one or both ssDNA strands has a single nicking site adjacent to and distinct from the site(s) of foreign DNA insertion.
2) a nicking agent(s); and
3) an exonuclease or strand-displacing polymerase.
6.2) The cloning vector of (6.1) wherein the nicking site(s) is not a canonical or noncanonical restriction enzyme recognition sequence.
The references sited detail numerous methods applicable to carrying out the present invention. Indeed, an outstanding feature of the present invention is that essentially all of the methods required are exceptionally well known within the molecular biological literature. So as to concisely and clearly disclose the present invention, rigorous duplication of these methods has not been done.
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
6063604 May 16, 2000 Wick et al 435/91.2. "Target nucleic acid sequence amplification"
5916777 June 29, 1999 Kacian et al 435/91.1 "Enzymatic synthesis of oligonucleotides using 3'-ribonucleotide primers"
5824517 Oct 20, 1998 Cleuziat et al 435/91.2. "Method for amplifying nucleic acid sequences by strand displacement using DNA/RNA chimeric primers"
5712124 Jan 27, 1998 Walker et al 435/91.2. "Strand displacement amplification"
5650271 July 22, 1997 Richards et al 435/6. "Enzymatic synthesis of oligonucleotides"
5648211 July 15, 1997 Fraiser et al 435/6. "Strand displacement amplification using thermophilic enzymes"
5270184 Dec 14, 1993 Walker et al. 435/91.2. "Nucleic acid target generation"
Alves et al "Accuracy of the EcoRV restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences" Biochemistry 1995 Sep 5;34(35):11191-11197
Bekkaoui et al "Cycling probe technology with RNase H attached to an oligonucleotide" Biotechniques 1996 Feb;20(2):240-8
Bishop et al "Plasmid cloning vectors that can be nicked at a unique site" Mol Gen Genet 1980;179(3):573-80
Champoux et al "Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase" J Virol 1984 Mar;49(3):686-91
Christ et al "The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate" EMBO J 1999 Dec 15;18(24):6908-16
Christensen et al "Target specificity of the endonuclease from the Xenopus laevis non-long terminal repeat retrotransposon, Tx1L" Mol Cell Biol 2000 Feb;20(4):1219-1226
Hancox et al "Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease" Biochemistry 1997 Jun 17;36(24):7577-85
Kainz et al "A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes" Anal Biochem 1989 Jun;179(2):366-70
Karwan et al " Yeast ribonuclease H(70) cleaves RNA-DNA junctions" FEBS Lett 1986 Oct 6;206(2):189-92
Lanio et al "EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides" Protein Eng 1996 Nov;9(11):1005-1010
Marshall et al "The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene" Nucleic Acids Res 1992 Dec 11;20(23):6401-6407
Parks et al "Phosphatidylcholine and phosphatidylethanolamine enhance the activity of the mammalian mitochondrial endonuclease in vitro" J Biol Chem 1990 Feb 25;265(6):3436-9
Pendergrast et al "High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates" Science 1994 Aug 12;265(5174):959-62
Saba "Strand Displacement Amplification 'SDA': A potentially constant temperature, exponential-like nucleic acid amplification process" Icosascan 1991 Nov 13; 4(23):30
Sheflian et al "Hydrolysis of DNA-duplexes, containing 5-fluorodeoxycytidine by restriction endonucleases" [Article in Russian] Biokhimiia 1993 Nov;58(11):1806-11
Venditti et al "A DNA conformational alteration induced by a neighboring oligopurine tract on GAATTA enables nicking by EcoRI" J Biol Chem 1991 Sep 5;266(25):16786-16790
Voigt, et al "O6-methylguanine in place of guanine causes asymmetric single-strand cleavage of DNA by some restriction enzymes" Biochemistry 1990 Feb 13;29(6):1632-7
Wende et al "The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV" Biol Chem 1996 Oct;377(10):625-32
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Sequencing
1) Nucleotide Sequence Variation AnalysesNucleotide Sequence Variation Analyses
Subtractive Primer Extension (SPE)
Mismatch Cleavage Assays (MCA)
2) Sequencing by Stepped Nucleotide Addition (SSNA)
3) Sequencing by Stepped Oligonucleotide Addition (SSOA)
4) Nested Primer Sequencing (NPS), with Universal Base-containing Primers
Nested Primer Sequencing by nucleotide extension (NPSnt)
Nested Primer Sequencing by oligonucleotide ligation (NPSo)
The first major embodiment of the present invention involves the analysis of nucleotide sequence variations, such as nucleotide transversion, transitions, deletions, insertions, and lesions.
Subtractive Primer Extension (SPE)
The first of these analyses is termed Subtractive Primer Extension (SPE),
which is similar to, yet patentably distinct from minisequencing and its
numerous related variations (1-26). In general these prior processes involve
hybridizing a primer to a target such that its 3' nucleotide is either
adjacent to, of overlaps the target nucleotide being interrogated.
Subsequently, extension with one or more labeled nucleotides, respectively,
under the appropriate conditions results in primer extension of only those
primers which are perfectly hybridized.
In SPE is distinct from most forms of minisequencing in that labeling is done, not via labeled chain-terminating nucleotides, but by multiple incorporations of labeled nucleotides onto those primers which have not been terminated by an unlabeled chain terminating nucleotide.
Figure #10 delineates the essential characteristics of the process one variation of this process wherein the primer's 3' end is adjacent to the target nucleotide being interrogated. The primer (or probe) and target sequences in this and most if not all processes thought out the present application can be either RNA, DNA or any variations thereof. The primer (or probe) in these processes is particularly amenable to derivation, and can contain any modifications the myriad available which is conducive to the particular process. Futher, the primer (or probe) need not be affixed, and could have separation tag (biotin).
In the example of Figure #10, four essentially identical separate, or separated support-affixed primers (thick lines) are shown hybridized to an identical target single stranded nucleic acid (thin lines). In practice, preferably there are multiple sets of distinct sequence identical primers affixed to separate or separated support surfaces. Each interrogating a distinct nucleotide within one or more targets.
The target nucleotide immediately adjacent to the primer's 3' terminus (shown here as G) is that which is being analyzed. In practice we do not know, and are attempting to identify this nucleotide.
In the first step, each separate, or separated primer-target hybrids are subjected to one unlabeled terminating nucleotide under primer extension conditions. The particular type (e.g. dideoxy) is not critical, but preferably incorporation is highly efficient and base-pair specific.
The next step is to provide the complete array with an excess of polymerizable nucleotides, at least one type of which is labeled "*". An advantage of SPE is that more than one label can be incorporated during primer extension.
Upon detection, the presence of a labeled primer indicates that the target nucleotide adjacent to the 3' end of the primer was not complementary to the chain terminating nucleotide used in the first step. In other words, the absence of label indicates the target nucleotide was complementary to the particular chain terminator used in the first step.
Primer extension with labeled nucleotides in the second step need not be achieved via a polymerase. For example, a terminal transferase could be used to multiply extend and label nonterminated primers, with or without the presence of target.
Although preferably probes are affixed and perhaps arrayed to a support, this need not be so and reactions can be done in solution. For example, primers could have universal addressing 5' tails (31,32). This alternative of doing reactions in solution is also applicable to many if not all processes of the present application.
In the example in Figure #10 the exact identity of the target nucleotide is determined. However, if simply determining if a particular target nucleotide is present or absent, then only one primer is necessary. This ability to reduce the number of primers needed when the exact identity of the target nucleotide is not required is generally applicable to most if not all the sequence variation analyses of the present invention.
The term "label" used above and thought out this application is, unless otherwise indicated, taken to mean any molecule or macromolecule that by itself, or after processing, results in a detectable signal. A considerable number of such labeling-detection schemes are well known to those skilled in the art. With certain processes of the present invention, as exemplified with SPE, the "label" of the labeled nucleotide need not result from an unnatural nucleotide structure. For example, if the target is DNA, and the nucleotides used in primer extension are ribonucleotides, a 2'-hydroxyl could be considered a label, wherein the resultant DNA-RNA hybrid is detectable with antibodies (33).
A particularly interesting form of labeling is that which can be detected electronically, via an electronic "biochip" (see Nat Biotechnol 2000 Oct;18(10):1096-100; Nucleic Acids Res 1999 Dec 15;27(24):4830-7 ;Science 1999 Jan 15;283(5400):375-81, and US Patent# 6,177,250. For example, this can occur by electron transfer via the nucleic acid from a terminal label to the chip surface. In another example, a mismatch can alter the conductivity of a modified or unmodified nucleic acid.
One form of describing SPE is that it is a process of sequence analysis or sequencing, via primer extension, which involves;
i) hybridizing a support affixed primer to a nucleic acid target such that the 3' end of the primer is immediately adjacent to the nucleotide of target being analyzed or sequenced;Figure #11 exemplifies variations of SPE wherein the target nucleotide in question is interrogated by hybridization with the last 3' nucleotide of probe. After extension of only the perfectly hybridized probe with a chain terminating nucleotide the remaining 3' mismatch complexes are subjected to various means of extension. As shown, one of the means of extending the nonterminated primers in this figure is by first cleaving the mismatched terminal nucleotide. Several other variations on this theme are presented in the next section. In this sequence variation analysis, and in those that follow, the primers need not be separate or separated, and all four chain terminating nucleotides can be simultaneously presented to all primer-target hybrids.
ii) providing polymerase and a base-distinct chain-terminating nucleotide such that if the target nucleotide adjacent to the 3' end of the primer is complementary to said base-distinct chain-terminator, then incorporation of this chain-terminator occurs;
iii) subsequently providing polymerizable nucleotides at least one kind of which is labeled, sufficient to extend and label a nonterminated primer.
Mismatch Cleavage Assay (MCA) The sequence variation analyses as exemplified by Figures #12-#17 involve primers or probes which contain 3' terminal or internal nucleotides which overlap and interrogate the target nucleotide variation in question. Those primers or probes which are not perfectly hybridized are cleaved at the mismatched nucleotides. Detection is by labeling either post cleavage (Figures #12 and #13), or by loss of label from prelabeled probes (Figures #14-#17).
Figure #12 shows the first derivation of MCA wherein chain terminating nucleotides are already positioned at the 3' ends of the nonlabeled probes (i.e., latent primers) before hybridization, thus avoiding the step of chain termination, and avoiding the need of having separated primers as in Figure #10. With cleavage and removal of the mismatched terminators, followed by various means of extension with one or more labeled nucleotides, those primers whose terminal nucleotides are not perfectly hybridized with the the target nucleotide in question are detectably labeled.
The mismatch cleavages in this and the following figures can be achieved by physical (e.g., light), chemical and/or enzymatic means (34-62). It may be advantageous to use more then one agent simultaneously, such as a glycosylase and apurinic/apyrimidinic (AP) endonuclease.
The process in Figure #13 is as Figure #12 except that the primer's nucleotide which is interrogating the target is within the probe (i.e., latent primer) rather than at the terminus of the primer. These probes are also specialized in that they have 3' terminal "cap" (e.g., dideoxynucleotide) which prevents their extension if they are not cleaved. Depending on the method and precise location of probe cleavage, various means of extension can be employed, optionally with removal of target sequences which may or may not have also been cleaved.
Figures #14 and #15, as #12 and #13, involve mismatch cleavage of terminal and internal mismatches, respectively. However, the probes in these process are terminally prelabeled before hybridization and mismatch cleavage, and it is the loss of label with cleavage that defines the positions of the mismatches. Preferably there are multiple labels at each termini. If target is not cleaved or stripped away after cleavage, then labeled 3' terminal fragments need be small enough that they dissociate from targets.
A variation of #15 which allows for detecting and locating any mismatch, deletion or insertion within a given length of target sequence involves ligation of an oligonucleotide (oligo) to the cleavage terminus of the probe. Herein the probe sequence is a "standard" full length complement of the target sequence to be scanned for variation. Either the probe or the target is selectively cleaved, or if both probe and target are cleaved new target could be hybridized to cleaved probes. The oligo ligated indicates the location within the probe (and target) at which the mismatch, deletion or insertion occurs. Yet another variation of this is to do chain terminating sequencing using the cleaved probe as primer and uncleaved or fresh target as template.
Figures #16 and #17 also involve probes which are prelabeled, but here the labeling is within the probes, not at the termini as in Figures #14 and #15. The assays in Figures #7 and #8 involve the use of end "caps" so as to protect them from exonuclease (63). After selective extension or mismatch cleavage, 5' and/or 3' exonuclease digests those which have been extended or cleaved thus liberating their labels. A variation on this theme, but without the need for end caps, is to first bind and protect those labeled mismatched probes with MutS protein and subsequently digest those nonbound labeled probes with exonuclease (64).
Figure #18 is unique in that it is the target and not the primer or probe which is labeled. The mismatch nucleotide is positioned in the probe such that after mismatch cleavage of target and/or probe, the cleaved target fragments are capable of being washed away. Labeling of target can be done by a variety of means as well known by those skilled in the art.
Since primer extension is not involved in the assays of Figures #14, #15, #16 or #17 the probes can be affixed to the support either by their 5' or 3' ends.
The above MCA processes wherein the target interrogation is done with internal probe sequences are applicable to sequence variations other than single nucleotide polymorphisms, such as insertion, deletions, and lesions.
Since some mismatch cleavage methods require analysis of the sequences present on both strands of target, the pair of correspondingly hybridizing probes may be required.
Some of the above descirbed assays may be accomplished in solution, such as utilizing a separation modification (e.g. biotin) to the probes/primers.
Finally, mismatches can be detected as a result of being recognized by proteins such as MutS, or by specially designed and mismatch-selective intercalaters.
Sequencing via Stepped Nucleotide Addition (SSNA)
A form of sequencing which does not require electrophoresis involves the
sequential addition of fluorescently-labeled reversibly-blocked 3'-blocked
nucleotide (65-69). Analogous to minisequencing, subsequent to addition of
one nucleotide to a primer hybridized to the target, this nucleotide's
distinct label allows its identification. Sequential deblocking, extension
with labeled reversibly-blocked nucleotides, and detection provides the
target sequence.
Although at first glance this novel means of sequencing seems quite promising, it has yet to become practical. One significant source of difficulty appears to be the lack of completeness of the reactions performed, which is a rapid loss of signal-to-noise.
The next embodiment of the present invention "Sequencing via Stepped Nucleotide Addition" (SSNA) was conceived to, among other things, help alleviate this loss of signal-to-noise due to reaction incompleteness.
The essential distinctions of SSNA relative to known base-addition sequencing schemes include:
1) reversibly-blocked labeled nucleotides are not employed, and the cycled addition and delabeling of labeled nucleotides to the same primer is not involved;The phase "essentially identical" is meant clarify that a "primer" or "target" need not be polynucleotides identical in all respects. For example primers of slightly different length at their 5' ends, but whose critical 3' target-hybridizing sequences are identical, would be "essentially identical". This also applies to the target, wherein sequences flanking the primer binding site and the downstream sequences in question, can be different in length without causing the targets to be "essentially identical".
3) multiple initially essentially identical primers, hybridized to essentially identical targets, are differentially extended, and subsequently are labeled only once.
SSNA could be generalized as follows.
(A) A process for determining the sequence of a polynucleotide, comprising:Importantly, an advantage of using of arrayed primers, rather than "batch" solution reactions, is that the process can be done in multiplex fashion. That is at each section of the array more than one primer can be positioned, which can be directed to distinct target sequences, present on the same or distinct target nucleic acid fragments.i) hybridizing multiple essentially identical primers to multiple essentially identical targets such that the 3' ends of the primers are positioned at the same location within the targets;Figure #19 exemplifies this process using a "batch" solution reaction from which small sections are extracted with each cycle. As an aid in understanding this and other figures, descriptive text has been incorporated which may not be needlessly duplicated in the text.
ii) segregating a relatively small section of these primer-target hybrids;
iii) subjecting the remaining primer-hybrid complexes to polymerase and nonlabeled reversibly-blocked chain-terminating nucleotides such that all of the primers are extended by one nucleotide, the identity of which is templated by the target sequence;
iv) optionally "capping" those unextended 3' termini, perhaps with more readily incorporated chain-terminating nucleotides under conditions promoting relaxed (error prone) nucleotide incorporation by the polymerase;
v) removing or disabling uncoupled reversibly-blocked chain-terminating nucleotides,
vi) removing, optionally concurrent with (v), the reversible block from the primer extension product of (iii);
(v) repeating steps (ii) through (vi) multiple times, such that at the end thereof there are an array of segregated small sections, each section containing a distinctly lengthed primer.An alternative to this process is to perform the small section segregations when the primers are capped with the 3' reversibly-blocked chain-terminating nucleotide, rather than the a free 3'-OH as defined above. This alternative is exemplified in Figure #20 where SSNA is achieved using arrayed primers.
SSNA as applied to arrayed primers can be summarized as follows.
(B) A process of determining the sequence of a one ore more target nucleic acids, comprising:
i) to each of multiple identical sections of a support, positionally arraying primers of the same or distinct sequence, and whose 3' ends are reversibly-blocked so as not to allow for extension,(C) The process of (B) wherein after all rounds are completed all of the 3' ends are deblocked and extended by one labeled chain-terminating nucleotide, the identity of which is templated by the target sequence.
ii) hybridizing one or more targets to said arrayed primers,
iii) selectively deblocking the 3' ends of all but one section of primers,
iv) providing polymerase and reversibly 3' blocked chain-terminating nucleotides so as to extend all those primers which have been deblocked in step (iii).
(vi) repeating steps (iii) and (iv) such that with each cycle one more section is added to those protected from deblocking, and the sizes of the primers from one section to the next is increased by one nucleotide.
Perhaps as important as reducing the complexity of the reactions which are required in the prior processes of nucleotide addition sequencing, is the ability to label all of the arrayed sections only once and at one time. This allows SSNA much greater flexibility in designing labeling and detection schemes. For example, photocleavable mass labeled chain-terminating nucleotides could be used, followed by mass spectrometry. Another example would be to use derivatized chain terminating nucleotides (e.g., ddUTP-biotin), which allow for well known enzymatic colorimetric techniques, using such reagents as avidin-coupled alkaline phosphatase or antibody-coupled peroxidase.
A particularly interesting form of labeling is that which can be detected electronically, via an electronic "biochip" (see Nat Biotechnol 2000 Oct;18(10):1096-100; Nucleic Acids Res 1999 Dec 15;27(24):4830-7 ;Science 1999 Jan 15;283(5400):375-81, and US Patent# 6,177,250. For example, this can occur by electron transfer via the nucleic acid from a terminal label to the chip surface. In another example, a mismatch can alter the conductivity of a modified or unmodified nucleic acid.
It is very important to recognize that because labeling is effected at one time, some the previously disclosed Subtractive Primer Extension (SPE) and Mismatch Cleavage Assay (MCA) processes could also be applied to SSNA; and conceivable also to certain of the remaining sequencing processes to be described.
Sequencing by Stepped Oligonucleotide Addition (SSOA)
Important derivations of SSNA involve ligation of oligonucleotides rather
then polymerase incorporation of nucleotides. That is the individual stepped
additions are with reversibly-blocked oligonucleotides rather nucleotides.
Alternatively if an appropriate ligase was used, instead of a reversibly
blocking the oligo reagents, a ligated oligo with a 5' hydroxyl group could
be activated for further extension by 5' phosphorylation.
As with step wise addition and detection of reversibly labeled nucleotides, so too has step wise addition and labeled oligonucleotides been described (70-75).
However, just as SSNA is distinct from the previous disclosed process of step wise nucleotide addition, so too "Sequencing by Stepped Oligonucleotide Addition" (SSOA) only performs labeling and detection at the final step after all cycles of addition have occurred.
While SSNA requires only the 4 nucleotides, use of oligos requires a larger set of distinct sequence addition reagents. For example, for tetramers, 256 distinct oligos are required to represent all possible tetramer sequences in target and provide for complete sequence determination. For pentamers the number of required oligos grows to 1024. For less comprehensive sequencing, less oligonucleotides containing universal bases could be envisioned. (76-79).
Because primer extension via ligation can occur in either the 5' or 3' direction, support affixed primers can be attached to the support either by their 3' or 5' end. Further, both enzymatic (e.g., T4, T7, Pfu, Taq, or E. coli ligase) and chemical ligation (80-82) are applicable.
At the last labeling step, if tetramers are used, then 256 distinct labels, or label combinations, would be needed. To aid in the stabilization of hybrids formed from small labeled oligos, such as tetramers and petamers, one or more universal bases to be incorporated in the oligos. For example a tail could be positioned at the end of the oligo which is not adjacent to the primer. This could also facilitate labeling in that the labels could be positioned on the universal nucleotides within this tail, and thus would be less likely to interfer with ligation.
As a final note, if stacking interactions can sufficiently secure the final labeled oligo in place, and stacking of multiple labeled oligos on a target can be prevented, then ligation may need not be done. This is also applicable to the next oligo-based version of Nested Primer Sequencing (NPS) described in next section of this application.
Nested Primer Sequencing (NPS), with Universal Base-containing Primers
(Nucleotide (NPSnt) and Oligo (NPSo) variations)
This embodiment of the present invention involves a highly novel means of
DNA or RNA nucleic acid sequencing which avoids the need for multiple
manipulations and gel electrophoresis of all currently known nucleic acid
sequencing processes.
More specifically this invention involves a process of sequencing a target nucleic acid (ss or ds DNA or RNA) using a nested set of support arrayed, universal base-containing polynucleotide primers. As with all the processes of the present invention the target and primer (or probe) can be RNA and/or DNA, or derivations thereof. As seen in Figure #21 these primers also contain a known "registering" sequence whose complement is present within the target to be sequenced, and which precisely aligns the universal base containing sections of the primers in their hybridization to the target.
Universal nucleotides base-pair with any of the naturally occurring bases (adenine, guanine, cytosine, or thymine (uridine)). Examples of such universal bases would include, but not be limited to, 5-nitroindole, 3-nitropyrrole, inosine, pyrrole-3-carboxamide and imidazole-4-carboxamide (83-103). Universal bases which predominately are stabilized by stacking rather than hydrogen bonds, such as those composed of aromatic hydrocarbons and heterocycles (80) may find utility. More than one type of universal base could be incorporated into a single primer.
Use of universal bases in sequencing by arrays has been reported. For example there have been reports using probes that contain several universal bases at both ends(76,77). There has also been reports of using the universal bases to reduce the number of oligonucleotides used in a sequencing by hybridization scheme (78,79).
Use of a common known registering sequence at one end of arrayed probes is also known (104). Therein this sequence is directly connected to a second part whose sequences consist of permutations of 2 to 6 of the natural nucleotides, A, T, G or C. A similar array of binary probes is claimed by Chetverin, et al (104.1).
As shown in the Figure #21, the polynucleotide fragments arrayed are precisely distinct from each other by a given number of universal base-containing nucleotides, in this example 1 such universal nucleotide. This number can be larger, since sequential base-addition sequencing (65-69) or Sequencing by Stepped Nucleotide Addition (SSNA) could be used to fill in the gaps.
Although for clarity this example only shows 4 arrayed primers, which are distinct in length from the next by 1 universal nucleotide, there would preferably be more of these distinctly lengthed primers so as to sequence longer sections of target. The lengths of the steps of universal base sequences within the primers will depend on the length of the target to be sequenced. For example if 250 nucleotides of the target are to be sequenced, and the arrayed primers are distinct by a step of 1 universal nucleotide, then there would be 250 distinctly lengthed primers.
While in the figure only one of each fragment length is exemplified, it is necessary that a sufficient number of identically lengthed primers be positioned at the same place in the array so as to generate a detectable signal.
The sequence and length of the registering sequence can vary. However, the Tm of the hybridized aligning sequencing must be sufficient to overcome the relatively nonspecific competing hybridizations among the target and the universal sequences, and perhaps the hybridizations of the universal sequences among themselves. Besides simply increasing the length of the registering sequence, there are several known means of achieving primer stabilization. For example base derivations such as 5-methylcytosine are know to increase hybrid stability. Other means are though polynucleotide backbone modifications such as Peptide Nucleic Acid (PNA) (105). Another possibility is conjugating a minor groove binding stabilizer (106).
Yet another particularly interesting means of increasing hybrid stability are via covalent coupling of a modified oligonucleotide to its target, triggered upon hybridization (107,108). The temperature and stringency of hybridizing the targets to the arrayed polynucleotides will depend on among other things, the exact buffer composition and conditions of hybridization, lengths of the targets to be sequenced, the type of universal base(s) used, and the type and length of the registering sequence. The slope of the temperature drop during hybridization could also be important.
One of the versatile aspects of the present invention is that sequencing can be done in multiplex, wherein several distinct targets, or several distinct target sections of one polynucleotide, are simultaneously sequenced. In this case each distinct target sequence will have a distinct register-complement sequence which hybridizes to a distinct registering sequence present in a subset of the primers. Note that the register-complement sequence in target need not be know in advance, for example is could be an octamer sequence.
Continuing with Figure #21, subsequent to aligning the target, primer extension by one nucleotide using distinctly labeled chain terminating nucleotides which contain the natural bases is effected. It is of course important that the 3' universal nucleotide function as a substrate for primer extension, accurately incorporating that terminating nucleotide which is complementary to the target nucleotide just past the 3' of the primer. Alternatively, if a particular universal nucleotide is found difficult to extend,each distinctly sized primer could be a mixture of four wherein their 3' ends contain either A, T, G, or C. Only one of these 4 "capped" species will be extended by the labeled terminating nucleotide do the others be mismatched. Note the potential of employing SPE or MCA.
Subsequently to "washing" of uncoupled nucleotides, and perhaps removing the targets by a denaturation "washing", the label of the 3' coupled nucleotide of each distinctly sized primer is determined . Thus revealing the identity of the 3' coupled nucleotide, and objectively the complement nucleotide of target.
As a final note, Figure #21 exemplifies the use of a "spacer" which can be any suitable noninterfering linkage which aids in the hybridization step. See Southern, et al (109) for examples of this and general discussion of chip hybridization technology.
Figure #22 is as #21 except that at the ends of the nested universal primers is one of four natural nucleotides which interrogates the target nucleotide in question by overlapping it. This is analogous to SPE as shown in Figure #11 & #12, and also MCA as shown in Figure #14.
Figure #23 is a derivation of NPS, called Scanning Mismatch Sequencing (SMS) where instead of having distinctly sized universal base-containing primers, all of the probes (or primers) contain the same number of universal nucleotides, and are distinct as to the position of the natural nucleotide interrogating the target. That the probes are the same length could ad in uniform hybridization of target(s) to probes. Note several of the derivations of labeling seen above in MCA are applicable here.
A particularly interesting form of labeling is that which can be detected electronically, via an electronic "biochip" (see Nat Biotechnol 2000 Oct;18(10):1096-100; Nucleic Acids Res 1999 Dec 15;27(24):4830-7 ;Science 1999 Jan 15;283(5400):375-81, and US Patent# 6,177,250. For example, this can occur by electron transfer via the nucleic acid from a terminal label to the chip surface. In another example, a mismatch can alter the conductivity of a modified or unmodified nucleic acid.
The oligo ligation version of NPS (NPSo)is delineated in Figure #24. NPSo is related to the the above nucleotide version of NPS (NPSnt) just as SSOA is related to SSNA, and most of that which was said in the above description of SSOA is applicable to NPSo. The labeled oligos used in NPSo and SSOA, in addition to containing natural nucleotides, could also contain one or more universal nucleotides variously positioned. This modification could be particularly valuable in labeling.
References
The prior art referenced throughout this application and listed below are extensive and valuable sources of proven applicable procedures for carrying out the processes of the present invention. Although for clarity of disclosing the present invention, these procedures are not rigorously duplicated in the text of this provisional application, they are assumed to be a component.
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Differentially Reactive Labels
The phrase "reactive label" indicates that which by physical, chemical or
enzymatic means are disabled or enabled to result in the absence or
appearance of a detectable signal, respectively; or liberated from their
conjugated molecule (often times a probe).
In both SSOA and NPSo, the large number of distinct sequence oligonucleotides provides a special problem in labeling. Mass tags have been found especially well suited for such labeling (70,110).
Use of photocleavage labels alone or in combination is well known (70, 110-117). The mass labeling processes of this embodiment of present invention are distinct from these in that the mass labels are linked directly to the monomer units of the polymer rather then via a tag moiety (70).
Modified nucleotides with photocleavable labels which would be useful. If for example we take the 256 tetramers, only 16 distinct mass labeled, placed in sets of 4 within the tetramers, or within an extension of the tetramer (e.g. tail of universal bases) would provide the necessary information required to determine the exact oligonucleotide sequence. That is, for each oligonucleotide there is provided 4 distinct labels, each of which is indicative of the position of the nucleotide within the tetramer. For pentamer oligos, 20 distinct labels are required; 5 for each of the 4 nucleotides, wherein one of said 5 identifies the location of that particular nucleotide within the pentamer.
However, and astonishingly so, this embodiment of the present invention is far more encompassing then photoliberatable mass labels, and includes novel processes, compounds, and libraries of compounds with labels reactive to various sorts of perturbations such as light, heat, enzymes, or chemical(s). Furthermore, reactive and nonreactive labels can be combined to allow even greater combinatorial coding capacity with a given number of labels.
Further description of this embodiment of the present invention are perhaps most clearly and concisely described in provisional claim format as follows:
A) A molecule with multiple (affixed) labels wherein one of the labels is selectively reactive (such that upon reaction this selectively reactive label is disabled, enabled, or liberated from the molecule [such that there is a detectable gain and/or loss of signal]).Alternative descriptions include:
B) A molecule containing multiple (affixed) labels wherein at least two of these labels are selectively and differentially reactive as in (1).
C) A molecule in (A) or (C) wherein the labels, before reaction(s), are distinct in their detectable signal.
1) A molecule which is multiply labeled and wherein these labels are differentially and detectably reactive such that there is a gain or loss of signal(s).Alternaitve descriptions include the following:
2) A molecule which is multiply labeled and wherein the linkages of these labels to the molecule are differentially cleavable.
3) A molecule of (2) wherein cleavage is effected light, heat, a chemical(s), and/or an enzyme.
4) A molecule of any of the above, wherein the molecule is a polymer, and wherein two or more monomers thereof are labeled.
5) A labeled molecule wherein the label is linked to the molecule via a linkage which is cleaved by an enzyme.
6) A labeled of molecule of (5) wherein the molecule if a nucleotide or derivation thereof, which may reside in a polynucleotide.
7) A labeled nucleotide, which may reside in a polynucleotide, wherein the label is conjugated to the nucleotide other than its 3' or 5' ends, and is cleaved from the nucleotide by an enzyme or chemical.
8) A library composed of distinct species of any of the above.
9) A process for combinatorial labeling of a distinct sequence polymer which is a member of a multitude of distinct sequence polymers, comprising labeling each of multiple monomers of said distinct sequence polymer with a reactive or nonreactive label, the combination of which defines the sequence of the distinct sequence polymer.
10) A process for combinatorial labeling of a distinct sequence polymer which is a member of a multitude of distinct sequence polymers, comprising labeling each of multiple monomers of said distinct sequence polymer with a photoliberatable mass label, the combination of which as detected by mass spectrometry defines the sequence of said distinct sequence polymer.
11) A process of 9 or 10 wherein the polymer is a polynucleotide or derivation thereof.
(A) A labeled polynucleotide, or derivative thereof, wherein their are at least two distinct labels linked via a photocleavable linkages.Figure #25 provides a general outline of utilizing a relatively small number of differential reactive labels to label a multitude of identities.
(B) A labeled terminating nucleotide triphosphate, or derivative thereof, wherein the label is linked via a photocleavable linkage.
(C) The labeld compound in (A) or (B) wherein the labels include a fluorscent label.
(D) The labeld compound in (A) or (B) wherein the labels include a mass label.
(E) A labeled nucleoside, nucleotide, polynucleotide, or derivative thereof, wherein the label is linked via a photocleavable linkage which does not contain a phenyl substituted with a nitro, sulfoxide, alkyl, or alkoxyl group.
(F) A labeled nucleotide triphosphate as defined in (E) which functions as a chain-terminator.
(G) A labeled nucleotide triphosphate as defined in (E) which can be incorporated multiple times during extension of a polynucleotide.
(H) A labeled polynucleotide as defined in (E).
(I) A labeled polynucleotide of (H) which functions as a primer in nucleic acid sequencing.
(J) A labeled polynucleotide of (H) or (J) which has multiple labels.
(K) A labeled polynucleotide of (J) wherein the labels are distinct.
(L) Any of the above wherein the label is a mass label, a fluorescent label, a solid support, a chemoluminescent label, a peptide, or a protein.
(M) Any of the above wherein the label is a fluorescent label.
Differential reactive labels are of particular utilty in a process termed Multiplex Electrophoresis. Particularly, Multiplex Electrophoresis is applicable to the simultaneous electrophoretic separation and detection, on one gel lane, of the chain terminated fragments of two or more sequencing templates, as shown in Figure #26.
The essential characteristic of ME is that the variably lengthed polynucleotide fragments of one set are selectively disabled, enabled, or removed from those of another set during electrophoresis.
The currently most efficient means of sequencing is by electrophoretic separation of fluorescently-labeled chain terminated reaction products. Either the primer or chain terminating nucleotides are distinctly labeled so that the chain termination reaction products resulting from termination at thymidine (T), cytidine (C), guanosine (G) or adenosine (A) are identifiable when simultaneously electrophoresed.
Particularly important in this sequencing process is the fluorescent dyes used, and the means of their detection (30-40).
It is anticipated that with larger numbers of sufficiently distinguishable dyes it will be possible to simultaneously sequence the chain termination reaction products of more than one target simultaneously in one gel lane.
An example of an attempt toward this goal is Wiemann, et al (41) who teach use of different lasers to excite distinctly labeled termination products resulting in the simultaneous sequencing from the two strands of double-stranded templates. Detection is achieved online and in parallel.
A preferred embodiment of ME is distinct from this and similar attempts in that in contrast to increasing the number of detectable labels, it is based on the selective disabling of the chain termination reaction fragments of one target, from those of another target(s).
Figure #26 is a simple example of ME, wherein the base-specific chain termination reaction products of two target sequences are being simultaneously electrophoresed. For clarity only those reaction products terminating with a thymidine are used. Extrapolating the process to using four nucleotide base-distinct dyes can readily be envisioned. In essence, subsequent to the first combined reading (#26.1, #26.4) the labeled termination reaction products of one of the targets are selectively disabled (#26.2) such that during the second laser-induced fluorescence and reading (#26.3, #26.5) only the products of one of the targets remains. By subtracting the readout of this second laser-induced fluorescence from the readout of the first, the first target sequence is deduced.
Alternatively, instead of first reading the products of both targets, step #26.2 of Figure #26 could be enabling, rather than disabling, such that previously undetectable chain termination fragments subsequently become detectable (42-44).
As delineated in Figure #26 a preferred means of selective disabling is by an external electromagnetic radiation source. Most preferably disabling would involve destruction of the dyes. This could be achieved by affixing within, on, or around the dye a chemical group that upon photoactivation resulted in dye disabling. Such a photoactivatable group could be for example, a nitro, cyano, azido, or diazo.
Dependent on the dye, it may be fortuitously discovered that the radial producing photocleavable linkage is sufficiently close to certain dyes so as to effect their destruction in addition to their photoliberation.
In addition to intentionally affixing a disabling chemical group within, on, or around the dye; two dyes which have closely matched spectral properties may be found to be differentially inactivatable, such as by the process termed photobleaching (45). In such cases the energy imparted during the first laser excitation may be sufficient for selective photobleaching (disabling).
Another preferable means of disabling one target's reaction products would be to decouple the dyes away from the chain termination fragments. Chain terminating nucleotide derivatives such as the previously disclosed photocleavably labeled nucleotides could find application here. With regard to using photocleavable labeled primers, Olejnik et al (46-48) teach coupling various chemical groups, including fluorescent dyes, to the 5' end of oligonucleotides. Olejnik et al do not teach or suggest use of derivatized oligonucleotides as disclosed in the present invention.
It is important that if disabling is by photoliberation of the dye, then the photoreactive linkage not appreciably quench the dye (42). However, induced loss of such quenching could be as a means of enabling rather than disabling.
Decoupling is preferably done such that the labeled fragments do not interfere with either the previous or subsequent base readings. In designing the linkage it may be preferable that the dye-containing photoliberated fragments which are still fluorescent be neutrally charged and nonmigrating. Figure #27, exemplifies photocleavage of a labeled nucleotide to liberate a neutral, or near neutral dye containing fragment. Neutral BODIPY dyes (38) may be particularly applicable here. Alternatively, ionic groups could be incorporated into the photocleavable linkage so as to neutralize charged liberated dyes.
In certain circumstances it may be found that a photocleaved dye fragment even though charged, does not cause unacceptable interference with the first or subsequent reading.
Cleavage of dyes away from chain termination reactions fragments need not occur at the dye-nucleotide linkage. For example, cleavage of the dye labeled nucleotide, or its dye-labeled base, away from the chain termination reaction fragments would effect the desired disabling (49-52).
If 4 dyes are used in analysis of the chain termination fragments of two targets, then it may be preferable that the migration and fluorescence of the modified chain-termination reaction fragments not be appreciably altered by the presence of the modification. For example, if it was found that a a nitrobenzyl-containing linker altered migration of chain termination reaction products, one solution may be to likewise derivatize the nondisabled chain-termination reaction products with a nonphotoreactive chemical species similar in character to the reactive one (e.g., alkylbenzyl).
Preferably electromagnetic disabling occurs at a wavelength which does not appreciably alter the base-specific detection dyes. Likewise, the photodisabling or photocleavage moiety is preferably not responsive to the wave length of radiation used to excite the base-specific dyes.
Preferably also photocleavage is done so that the nondisabled chain termination reaction fragments are not unacceptably altered with respect to effect their migration.
Besides disabling or enabling chain termination reaction fragments, they could, at a precise location within the gel, be selectively arrested from further migration.
One means of achieving selective arrest of migration would be to affix a photo reactive chemical to the chain termination reaction products, which upon activation results in the crosslinking of these reaction products to the optionally derivatized gel matrix, thus halting their further migration. The selective incorporation of one or more photoreactive nucleoside derivatives as 5-iodo-2'-deoxyuridine or 5-azido-2'-deoxyuridine may be applicable (53-57). Several other examples, such sulfone (58) or diazo containing derivatives attached to the fragments, such as via the sequencing primer, may also find utility.
Alternatively the gel matrix itself could be derivatized with a photoreactive group which upon activation reacts with selectively modified chain termination reaction products of one of the targets. For example, the reaction products of one target could include sulfur-containing nucleotides (e.g., 6-thioguanosine, 4-thiouridine) so as to make them more reactive with a light-activated gel matrix (59-62).
Conceivably also, chemicals which are present in the gel matrix, either affixed to the polymer or free in solution, could ad in photodisabling, vaguely analogous to the fluorescent energy transfer.
Selective disabling, enabling, or arrest need not require induction from an external source, such as by radiation. That is, inherent in the gel at a defined position, there could be placed a substance which performs said selective disabling, enabling, or arrest.
For example, a reactive chemical, affixed to a defined position in the gel, could upon contact with a derivation on chain termination reaction fragments result in their disabling, enabling, or arrest.
Another possibility would be to specifically affix to a defined position in the gel, a "receptor" which binds to a "ligand" preaffixed to those chain termination reaction fragments to be arrested in their migration (63). The denaturing character of the gel buffer would need to be considered here.
With multiple distinct means of selective disabling or removal it would be possible to simultaneously sequence more than two target sequences.
In summary ME is a process for multiplex analysis of two or more polynucleotide fragment sets comprising disabling, enabling, or arresting the migration of at least one of the said sets during electrophoresis.
The apparatus for ME is also to be claimed.
References
The prior art referenced throughout this application and listed below are
extensive and valuable sources of proven applicable procedures for carrying
out the processes of the present invention. Although for clarity of
disclosing the present invention, these procedures are not rigorously
duplicated in the text of this provisional application, they are assumed to
be a component.
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