Methods for appending oligonucleotides directly to nucleic acid templates, particularly to defined sites internal to single-stranded templates, are described. Appending first and second common priming sites to each of a plurality of templates of distinct sequence allows the subsequent stoichiometric amplification of a plurality of templates of distinct sequence.

Description of the Invention

The present invention provides methods for appending one or more oligonucleotides directly to a single stranded nucleic acid template, typically (but not invariably) at one or more defined sites internal to the template. The oligonucleotides may be designed to provide one or more sites for priming the subsequent amplification of adjacent template regions. The methods may be readily multiplexed, permitting oligonucleotides to be appended, in a single reaction, to a plurality of templates. In multiplex embodiments in which the appended oligonucleotides provide one or more common priming sites, the plurality of templates may then be concurrently amplified using primers common to all templates. Such multiplexed amplification reactions provide high specificity and uniform amplification of all templates, solving problems that have plagued multiplex amplification reactions since the invention of PCR. 

In a first aspect, the invention provides a method of appending at least a first oligonucleotide directly to a nucleic acid template. 

The method may conveniently be understood by reference to the illustrative reaction of FIG. 1 (see Original Patent), in which oligonucleotide 20 is appended to a distinct site internal to template 10. 

In the first step of the method, illustrated in FIG. 1A (see Original Patent), template 10 and at least first oligonucleotide 20 are concurrently annealed to first probe 50. 

Probe 50 includes at least first template complementarity region 40 and at least first oligo positioning region 30 directly adjacent thereto: in the annealing step, template region 14 hybridizes to template complementarity region 40 of probe 50 and oligonucleotide region 22 concurrently hybridizes to oligo positioning region 30 of probe 50. 

The nucleotide of template complementarity region 40 and the nucleotide of oligo positioning region 30 that are directly adjacent within probe 50 define a junction within probe 50, and are hereinafter termed junctional nucleotides. In embodiments of the methods of the present invention in which two oligonucleotides are appended to template 10, as is illustrated in FIG. 2 (see Original Patent), first oligo positioning region 30a directly abuts template complementarity region 40 at a first probe junction, and second oligo positioning region 30b directly abuts template complementarity region 40 at a second probe junction. In such embodiments, the abutting nucleotides are thus respectively denominated first junctional nucleotides and second junctional nucleotides. 

Oligonucleotide 20 (synonymously, "oligo 20") includes terminal region 22 that is complementary in sequence to oligo positioning region 30 of probe 50. Oligo 20 may optionally include a further region 24. The terminal nucleotide of oligonucleotide region 22--in the orientation schematized in FIG. 1, the 5' terminal nucleotide of oligo 20--is annealed to the junctional nucleotide of probe 50's first oligo positioning region 30. 

Oligo 20 is typically further designed to include at least one sequence to which an oligonucleotide primer can later anneal (a "priming site"). 

In the next step of the method, a first ligatable free end is created in template 10 at the nucleotide of template region 14 that is annealed to the junctional nucleotide of template complementarity region 40 of probe 50. The template nucleotide at which the free end is created remains within template 10. 

The products resulting from this step are shown in FIG. 1B, illustrating that the newly created ligatable free end of template 10 and the terminal nucleotide of oligonucleotide 20 terminal region 22 are positioned directly adjacent to one another by concurrent hybridization to probe 50 regions 40 and 30, respectively. 

In a final step, first oligonucleotide 20 is ligated to the first ligatable free end of template 10, with results as schematized in FIG. 1C. 

In methods of the present invention template 10 is a single-stranded nucleic acid, typically DNA, and may be obtained by denaturation of double-stranded nucleic acids. 

Template 10 may, for example, be derived from cDNA. Template 10 may be derived directly from single-stranded cDNA, such as that obtained by first strand synthesis from mRNA transcripts, or from cDNA rendered single-stranded by denaturation of double-stranded cDNA obtained either directly after second strand cDNA synthesis or from prior-cloned double stranded cDNA. 

Template 10 may in other embodiments be derived from genomic DNA, either from a genomic DNA preparation prepared directly from cells or from genomic DNA that has been prior-cloned. Typically, genomic DNA is first denatured, e.g. by heat or treatment with base, to provide single stranded template 10. 

Template 10 may be derived from a single individual or pooled from a plurality of individuals. Templates from a single individual are useful, for example, in genotyping or haplotyping efforts, as may be practiced in molecular genetic diagnosis or prognosis. Pooled templates are useful in SNP discovery efforts, and may usefully be pooled from at least 10 individuals, 100 individuals, even 1000 individuals or more. 

Template 10 may usefully be derived from nucleic acids drawn from a prokaryote, eukaryote, or virus. 

Prokaryotes include both archea bacteria and eubacteria, including both gram negative and gram positive eubacteria, and the methods of the present invention find particular utility when template 10 is drawn from nucleic acids of pathogenic prokaryotes. 

Among eukaryotes, template 10 may usefully be drawn from protozoa, fungi, insects, plants, and animals, including fungi selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustillago maydis, Neurospora crassa and Candida albicans; insects selected from the group consisting of Drosophila melanogaster and Anopheles species; plants selected from the group consisting of experimental model plants such as Chlamydomonas reinhardtii, Physcomitrella patens, and Arabidopsis thaliana, crop plants such as cauliflower (Brassica oleracea), artichoke (Cynara scolymus), fruits such as apples (Malus, e.g. domesticus), mangoes (Mangifera, e.g. indica), banana (Musa, e.g. acuminata), berries (such as currant, Ribes, e.g. rubrum), kiwifruit (Actinidia, e.g. chinensis), grapes (Vitis, e.g. vinifera), bell peppers (Capsicum, e.g. annuum), cherries (such as the sweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), melons (Cucumis, e.g. melo), nuts (such as walnut, Juglans, e.g. regia; peanut, Arachis hypogeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica), pear (Pyra, e.g. communis), plum (Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata or vesca), tomato (Lycopersicon, e.g. esculentum); leaves and forage, such as alfalfa (Medicago, e.g. sativa or truncatula), cabbage (e.g. Brassica oleracea), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce (Lactuca, e.g. sativa), spinach (Spinacia, e.g. oleraceae), tobacco (Nicotiana, e.g. tabacum); roots, such as arrowroot (Maranta, e.g. arundinacea), beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota), cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g. rapa), radish (Raphanus, e.g. sativus), yam (Dioscorea, e.g. esculenta), sweet potato (Ipomoea batatas); seeds, including oilseeds, such as beans (Phaseolus, e.g. vulgaris), pea (Pisum, e.g. sativum), soybean (Glycine, e.g. max), cowpea (Vigna unguiculata), mothbean (Vigna aconitifolia), wheat (Triticum, e.g. aestivum), sorghum (Sorghum e.g. bicolor), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays), rice (Oryza, e.g. sativa), rapeseed (Brassica napus), millet (Panicum sp.), sunflower (Helianthus annuus), oats (Avena sativa), chickpea (Cicer, e.g. arietinum); tubers, such as kohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum) and the like; fiber and wood plants, such as flax (Linum e.g. usitatissimum), cotton (Gossypium e.g. hirsutum), pine (Pinus sp.), oak (Quercus sp.), eucalyptus (Eucalyptus sp.), and the like and ornamental plants such as turfgrass (Lolium, e.g. rigidum), petunia (Petunia, e.g. x hybrida), hyacinth (Hyacinthus orientalis), carnation (Dianthus e.g. caryophyllus), delphinium (Delphinium, e.g. ajacis), Job's tears (Coix lacryma-jobi), snapdragon (Antirrhinum majus), poppy (Papaver, e.g. nudicaule), lilac (Syringa, e.g. vulgaris), hydrangea (Hydrangea e.g. macrophylla), roses (including Gallicas, Albas, Damasks, Damask Perpetuals, Centifolias, Chinas, Teas and Hybrid Teas) and ornamental goldenrods (e.g. Solidago spp.); and animals selected from the group consisting of mammals, such as primates, including humans, monkeys, and apes, small laboratory animals, including rodents, such as mouse or rat, guinea pigs, lagomorphs, such as rabbits, livestock, such as cows, horses, chickens, geese, turkeys, goats, and sheep, and domestic pets, such as dogs and cats. 

Template 10 may also usefully be derived from viral nucleic acids, including viruses selected from the group consisting of double-stranded DNA viruses, such as herpes viruses, including human herpesvirus 1 and 2 (HSV-1 and HSV-2), varicella-zoster (HSV-3), cytomegalovirus (HCMV), human herpesvirus 6, 7, 8 (HHV-6, HHV-7, HHV-8), and Epstein-Barr virus (EBV), retroviruses, including mammalian type B retroviruses, such as mouse mammary tumor virus; mammalian type C retroviruses, such as murine leukemia virus and reticuloendotheliosis virus (strain T, A); avian type C retroviruses such as avian leukosis virus; type D retroviruses such as Mason-Pfizer monkey virus; BLV-HTLV retroviruses such as bovine leukemia virus; lentiviruses, such as bovine lentiviruses including bovine immunodeficiency virus, feline immunodeficiency virus, visna/maedi virus (strain 1514), and primate lentiviruses such as human immunodeficiency virus 1 (HIV1), human immunodeficiency virus 2 (HIV2), and simian immunodeficiency virus (SIV), and other types of pathogenic viruses. 

Template 10 is typically at least about 40 nt in length, often at least 50, 75, 100, 125 or 150 nt in length, at times at least about 200 nt, 300 nt, 400 nt, or 500 nt or more in length, and when derived from genomic nucleic acid can be at least 1000 nt (1 kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb or 50 kb in length. 

Probe 50 includes at least first template complementarity region 40. 

Template complementarity region 40 of probe 50 is designed to have sufficient length and sufficient sequence complementarity to a region of template 10 as to permit annealing of probe 50 to template 10 under hybridization conditions of desired stringency. The region of template 10 to which probe template complementary region 40 hybridizes is defined as template region 14. 

Template complementarity region 40 of probe 50 is typically at least 20 nt in length, more typically at least 35, 40, 45, or 50 nt in length, often at least 100 nt in length, 150 nt in length, even at least 200 nt, 300 nt, 400 nt, or even at least 500 nt or more in length. Template complementarity region 40 of probe 50 is typically no more than about 1000 nt in length, typically no more than about 500 nt in length, often no more than 400 nt, 300 nt, 200 nt, even no more than 100 nt in length. 

The length of template complementarity region 40 of probe 50 may further be chosen so as to hybridize to a region 14 of template 10 that includes the entirety of a template portion desired to be amplified. 

Template complementarity region 40 of probe 50 is further designed to have sufficient sequence complementarity to template region 14 as to permit annealing of probe 50 to template 10 under hybridization conditions of desired stringency. 

Template complementarity region 40 of probe 50 may, for example, be perfectly (that is, 100%) complementary in sequence to template region 14. Template complementarity region 40 may, in the alternative, be only at least 95% complementary, 90% complementary, 85% complementary, 80% complementary, even only at least 75% complementary in sequence to template region 14, with percent complementarity measured for the purposes of the present invention by the procedure of Tatiana et al., FEMS Mirobiol. Lett. 174:247-250 (1999), which procedure is effectuated by the computer program BLAST 2 SEQUENCES, available online at the National Center for Biotechnology Information (NCBI) website. 

With a pooled template, template complementarity region 40 of probe 50 may simultaneously have different degrees of sequence complementarity to various of the template regions 14 in the pooled template sample. 

Probe 50 further includes at least first oligo positioning region 30. 

First oligo positioning region 30 of probe 50 is typically at least about 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, even 16, 17, 18, 19, or 20 nt in length, and may be at least 25 nt, 30 nt, 40 nt, or even at least 50 nt in length or more. First oligo positioning region 30 of probe 50 is typically no more than about 50 nt in length, even more typically no more than about 40 nt in length, and may be no more than 35, 30, or even 25 nt in length. 

In the illustration of FIG. 1, first oligo positioning region 30 is positioned 5' to template complementarity region 40. This orientation is not required: first oligo positioning region 30 may be positioned 3' to template complementarity region 40. 

Probe 50 may be synthesized chemically, using solid phase procedures well known in the art, or by ligation of smaller, chemically-synthesized fragments. 

Typically, however, probe 50 is generated by first cloning the template complementarity region into a replicable vector, and then using flanking vector primers to amplify the sequence in a PCR reaction. To permit separation of template from probe after oligonucleotide ligation, the PCR reaction may usefully be performed using dUTP instead of dTTP; removal of uracil-containing probes from template is further described below. 

Oligonucleotide 20 is typically at least 15, 20, or 25 nt in length, and may be at least 30 nt, 35 nt, 40 nt, even at least 50 nt in length. Oligonucleotide 20 is typically no more than 50 nt in length, and often no more than 40, 30, even no more than 25 nt in length. 

Terminal region 22 of oligo 20 is designed to have sufficient length and sufficient sequence complementarity to probe region 30 as to permit annealing of oligo 20 to probe 50 under hybridization conditions of desired stringency. 

Terminal region 22 of oligo 20 is typically at least about 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, even 16, 17, 18, 19, or 20 nt in length, and may be at least 25 nt, 30 nt, 40 nt, or even at least 50 nt in length or more, and is typically no more than about 50 nt in length, even more typically no more than about 40 nt in length, and may be no more than 35, 30, or even 25 nt in length. 

Terminal region 22 of oligo 20 is further designed to have sufficient sequence complementarity to probe region 30 as to permit annealing of oligo 20 to probe 50 under hybridization conditions of desired stringency. 

Oligonucleotide terminal region 22 may, for example, be perfectly (that is, 100%) complementary in sequence to probe region 30. Oligo region 22 may, in the alternative, be only at least 95% complementary, 90% complementary, 85% complementary, 80% complementary, even only at least 75% complementary in sequence to probe region 30. 

Oligonucleotide 20 may optionally include a further region 24 that is noncomplementary in sequence to probe 50. This additional region 24 of oligonucleotide 20 may, for example, usefully include one or more restriction sites to facilitate subsequent cloning of portions of template 10 and may, in addition or in the alternative, include promoter sequences for phage RNA polymerases, such as T3, T7, and SP6 polymerases. 

Oligo 20 is typically further designed to include a sequence, a "priming site", to which an oligonucleotide primer can anneal in later steps. 

The priming site may be coextensive with either or both of terminal region 22 and optional region 24, or may be only a portion thereof. The length and sequence of the priming site sequence are chosen based upon considerations well known in the art, including the calculated Tm of the duplex expected between priming site and primer, the absence of the priming site sequence or its reverse complement in templates desired to be amplified, and the like. 

Annealing conditions for the first step of the methods of the present invention are chosen so as to permit concurrent annealing of template 10 and oligonucleotide 20 to probe 50. Typically, but not invariably, oligonucleotide terminal region 22 will be shorter than template region 14, and annealing conditions will thus be chosen principally to ensure that oligonucleotide 20 hybridizes to probe 50 with desired stringency. 

In the next step of the method, a first ligatable free end is created in template 10 at the nucleotide of template region 14 that is annealed to the junctional nucleotide of template complementarity region 40 of probe 50. 

In one series of embodiments, the ligatable free end is created by removing template regions that are noncomplementary to probe region 40, typically by treatment with nucleases active on single-stranded substrates. 

Usefully, ligatable free ends are created using exonucleolytic digestion, with the choice of nuclease determined by the desired direction (or directions) of template exonucleolytic digestion. For example, regions of template noncomplementarity to probe 50 may be removed by reaction with Exo I (3' to 5' exonuclease), Exo T (3' to 5'), rec J.sub.f (5' to 3'), Exo VII (both 3' to 5' and 5' to 3' exonuclease activity), and combinations thereof. 

In embodiments in which the only regions of template noncomplementarity to probe 50 are external to template region 14 (as in FIGS. 1 and 2, in contrast to FIG. 4 (see Original Patent), further described below), the ligatable free ends may also readily be created by use of an endonuclease, such as mung bean nuclease. 

In embodiments in which the free end is formed at the 5' end of template region 14, a further step of kinasing the template free end may be performed to ensure the presence of a 5' phosphate for subsequent ligation. 

First oligonucleotide 20 is then ligated to the newly created first ligatable free end of template 10 using a DNA ligase such as T4 ligase or, usefully, a thermostable ligase such as Taq ligase. Selection of ligase and ligation conditions are well within the skill in the art. 

The method for appending an oligonucleotide directly to a template, as schematized in FIG. 1 and described above, may be repeated, effecting the addition of a second oligonucleotide to the template. 

FIG. 2 shows an alternative series of embodiments in which first and second oligonucleotides 20a and 20b, respectively, are appended to template 10 in a single reaction. 

In these latter embodiments, probe 50 further includes second oligo positioning region 30b, additional to first oligo positioning region 30a. Second oligo positioning region 30b is directly adjacent to first template complementarity region 40. The nucleotide of template complementarity region 40 and the nucleotide of second oligo positioning region 30b that are directly adjacent within probe 50 define a second junction within probe 50, and are hereinafter termed second junctional nucleotides. 

Second oligo 20b includes terminal region 26 that is complementary in sequence to second oligo positioning region 30b of probe 50. 

Second oligo 20b may, like first oligo 20a, optionally include a further region 28 that is noncomplementary in sequence to probe 50. This additional region 28 of oligonucleotide 20 may, for example, usefully include one or more restriction sites to facilitate subsequent cloning of portions of template 10 and may, in addition or in the alternative, include promoter sequences for phage RNA polymerases, such as T3, T7, and SP6 polymerases. 

Second oligo 20b is typically further designed to include at least one site for subsequent priming of enzymatic polymerization. 

The priming site may be coextensive with either or both of terminal region 26 and optional region 28, or may be only a portion thereof. The length and sequence of the priming site sequence are chosen based upon considerations well known in the art, including the calculated Tm of the duplex expected between priming site and primer, the absence of the priming site sequence or its reverse complement in templates desired to be amplified, and the like. 

Template 10, first oligo 20a, and second oligo 20b are concurrently annealed to first probe 50. 

Template 10 anneals to probe 50 through hybridization of template region 14 to probe region 40, the template complementarity region. Terminal region 22 of first oligonucleotide 20a anneals to first oligo positioning region 30a, as in the embodiments above-described, and terminal region 26 of second oligo 20b anneals to second oligo positioning region 30b. The terminal nucleotide of first oligonucleotide terminal region 22 is annealed to the junctional nucleotide of the probe's first oligo positioning region. The terminal nucleotide of the second oligonucleotide's terminal region 26 is annealed to the junctional nucleotide of the probe's second oligo positioning region. 

In the next step, first and second ligatable free ends are created on template 10. The first ligatable free end is created in template 10 at the nucleotide of template region 14 that is annealed to the first junctional nucleotide of template complementarity region 40 of probe 50. The second ligatable free end is created in template 10 at the nucleotide of template region 14 that is annealed to the second junctional nucleotide of template complementarity region 40 of probe 50. The template nucleotides at which the free ends are created remain within the template. 

Thereafter, both first and second oligonucleotides are ligated to template 10: the first oligonucleotide to the first ligatable free end of template 10, the second oligonucleotide to the second ligatable free end of template 10. 

Usefully, ligatable free ends are created using bidirectional exonucleolytic digestion, either from a single bidirectional exonuclease or a combination of exonucleases having opposite directions of exonuclease activity. In embodiments in which the only regions of template noncomplementarity to probe 50 are external to template region 14 (as in FIGS. 1 and 2, in contrast to FIG. 4, further described below), the ligatable free ends may also readily be created by use of an endonuclease, such as mung bean nuclease. 

In another series of embodiments, illustrated in FIG. 3 (see Original Patent), first and second oligonucleotides are appended to template 10 in a single reaction using two probes, first probe 50a and second probe 50b. 

First probe 50a includes template complementarity region 40a and first oligo positioning region 30a. Second probe 50b includes template complementarity region 40b and second oligo positioning region 30b. Template 10 hybridizes to probes 50a and 50b through template regions 14a and 14b, respectively. 

The subset of these embodiments in which template regions 14a and 14b are not contiguous within template 10, as is illustrated in FIG. 3, usefully permit oligonucleotides to be appended to template 10 independently of the sequence intervening between template regions 14a and 14b, and thus permit oligonucleotides to be appended to template 10 despite variations that occur within the intervening region, such as exon insertions or deletions. This is represented in FIG. 3C by the plurality of hybridized template regions 10a and 10b. 

In such embodiments, exonuclease digestion, rather than endonuclease digestion, is typically used to create the first and second template ligatable free ends. 

FIG. 4 (see Original Patent) illustrates a further series of embodiments in which a single probe 50 is used to append first and second oligonucleotides to template 10. In contrast to the embodiments illustrated in FIG. 2, a region of noncomplementarity to probe interrupts template region 14. 

In such embodiments, exonuclease digestion may be used to create ligatable free ends in template 10, with results as schematized in FIG. 4B. Such embodiments usefully permit oligonucleotides to be appended to template 10 independently of the sequence interrupting template region 14, and thus permit oligonucleotides to be appended to template 10 despite variations such as exon insertions or deletions in this region. 

In alternative embodiments, endonuclease digestion, such as by mung bean nuclease, may be used to create first and second ligatable free ends, with results as schematized in FIG. 4C. In these embodiments, the endonuclease additionally removes the region of noncomplementarity that intervenes within template 10. 

These latter embodiments, in which endonuclease digestion is used to create ligatable free ends, permit templates that are perfectly matched to probe region 40 to be discriminated from templates that are imperfectly matched to probe region 40. In particular, when the method is followed by a step of bidirectional amplification, such as by PCR, only those templates that are perfectly matched to probe region 40 will be exponentially amplified; templates that are imperfectly matched will have been cleaved (as in FIG. 4C) and can at most be amplified unidirectionally, and thus geometrically. 

In a further series of embodiments, a priming site may be appended to template 10 without ligation of an oligonucleotide 20. In these embodiments, which require that a free ligatable 3' end be present or created in template 10, the priming site is appended to template 10 by DNA polymerase extension directly from template 10, rather than by ligation of a separate oligonucleotide species. In this reaction, the oligo positioning region, referred to as 30 in previous figures, a plurality of which is referred to here as 30a and 30b, of probe 50 acts as the "template" for directing enzymatic extension of template 10. 

In such embodiments, the first hybridization step is performed without addition of first or second oligonucleotides. After hybridization of template 50 to probe, referred to as 30 in previous figures, a plurality of which is referred to here as 30a and 30b, a DNA polymerase, such as Taq polymerase, is added to extend the template to append a priming site and, optionally, a molecular barcode (bar codes are further described herein below). Typically, this polymerization step is followed by a second hybridization step with the addition of an oligonucleotide 20, followed by ligation, as in the embodiments above-described. 

Any of the above-described embodiments may include a further step of separating template 10 from oligonucleotides 20 and probes 50. 

Typically, template 10 is readily separated from oligonucleotides 20 based upon size, using standard techniques such as gel electrophoresis, gel filtration, dialysis, or centrifugation in spin columns having size-selective membranes. 

In some embodiments, template 10 may also be separated from probes 50 based solely upon size differences. More typically, however, probe 50 includes means for separating the probe from template. 

For example, probe 50 may include deoxyuridine residues as substitutes for all or for a fraction of thymidine residues. 

In such embodiments, probe 50 may be treated after the ligation step with uracil-DNA glycosylase ("UDG"), which catalyzes the release of free uracil from uracil-containing DNA, creating apurinic ("AP") sites. 

AP sites may then be cleaved enzymatically using an AP endonuclease or, under certain conditions, an AP lyase. 

For example, the AP site may be cleaved using Endo IV or Fpg (formamidopyrimidine [fapy]-DNA glycosylase; also known as 8-oxoguanine DNA glycosylase). Fpg cleaves both 3' and 5' to the AP site, removing the AP site and leaving a 1 base gap. 

Alternatively, the AP sites may be cleaved chemically, such as by treatment with 1,4 diaminobutane and heat. 

In yet a further alternative, the probe can include purines such as 8-oxoguanine, 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil, that mimic damaged purines. Fpg glycosylase will release these residues from DNA and remove the resulting AP site, leaving a 1 nucleotide gap. 

The cleavage products of probe 50 may then readily be separated from template 10 by size separation, optionally with a preceding denaturation step. 

Probe 50 may instead, or in addition, include at least one capture moiety. The capture moiety is typically one member of a specific binding pair. 

"Specific binding" refers to the ability of two molecular species concurrently present in a heterogeneous (inhomogeneous) sample to bind to one another in preference to binding to other molecular species in the sample. Typically, a specific binding interaction will discriminate over adventitious binding interactions in the reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction is least about 10.sup.7 M.sup.-1, using at least 10.sup.8 M.sup.-1 to at least about 10.sup.9 M.sup.-1, and often greater, including affinities or avidities up to 10.sup.10 M.sup.-1 to 10.sup.12 M.sup.-1. 

The phrase "specific binding pair" refers to pairs of molecules, typically pairs of biomolecules, that exhibit specific binding. 

A wide range of specific binding pair members that can be used for capture of oligonucleotides are known in the art. 

Among these are small capture moieties colloquially termed "haptens" irrespective of their antigenicity. Such haptens include biotin, digoxigenin, and dinitrophenyl. Biotin can be captured using avidin, streptavidin, captavidin, neutravidin, or anti-biotin antibodies. Digoxigenin and dinitrophenyl can be captured using antibodies specific for the respective hapten. 

Capture of probe to a solid support, after ligation, permits separation of probe 50 from template 10 and oligonucleotides 20. 

In another series of embodiments, oligonucleotides 20 may include one or more capture moieties. In such embodiments, template 10 is typically first separated from unligated oligos by size separation, and template 10 then separated from probe 50 by capture of template molecules to which oligonucleotides have been successfully ligated. 

The methods of the present invention may include a further step, after ligating oligonucleotides to the template, of amplifying the template. Typically, the ligated template is first separated from probe and free oligonucleotides, and then amplified. 

As used herein, the term amplification includes the production of RNA transcripts by polymerization driven from a phage promoter. 

More typically, however, the amplification product is DNA produced by polymerization primed using one or more oligonucleotides ("primers") that are capable of hybridizing to one or more priming sites within one or more of the oligonucleotides appended to the template. 

For example, a first primer capable of binding to a first priming site present in the first oligonucleotide may be used to prime unidirectional amplification. A second primer capable of binding to the complement of the second priming site present in the second oligonucleotide may be used concurrently to prime bidirectional amplification. In embodiments in which first and second priming sites are reverse complements of one another, the first and second primers may be the same. 

Amplification may be isothermal or thermal cycling. 

Nucleic acid amplification methods useful in the methods of the present invention are well known in the art and include, e.g., polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), self-sustained sequence recognition (3SR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), rolling circle amplification (RCA), and strand displacement amplification (SDA). 

Typically, bidirectional amplification is effected using PCR. 

The methods of the present inventions may be readily multiplexed, permitting one or more oligonucleotides to be appended to a plurality of templates of distinct sequence in a single reaction. The templates may be separate nucleic acid molecules or separate loci of a single molecule such as a chromosome. In particularly useful multiplex embodiments, at least one, and typically two, common priming sites are appended to each of the plurality of templates. 

Thus, in another aspect, the present invention provides methods for appending at least a first oligonucleotide directly to a plurality of nucleic acid templates of distinct sequence in a single reaction. 

In such multiplex embodiments of the present invention, at least one oligonucleotide is appended to at least 2 templates of distinct sequence, typically at least 5 templates of distinct sequence, even at least 10, 20, 30, 40, or even at least 50 templates of distinct sequence, and may be appended to 100, 500, 1000, even 5000 or more templates of distinct sequence. 

In the first step, each of the plurality of templates of distinct sequence and a respective first oligonucleotide are concurrently annealed to a respective one of a plurality of probes within a single reaction mixture. 

As in the uniplex embodiments described above, each probe includes at least a first region of complementarity to a respective one of the templates and at least a first oligo positioning region directly adjacent thereto. Also as in the uniplex embodiments described above, the nucleotide of the template complementarity region and the nucleotide of the oligo positioning region that are directly adjacent within the probe are said to be first junctional nucleotides that define a first probe junction therebetween. 

Each first oligonucleotide includes a terminal region that is complementary to the first oligo positioning region of a respective probe. The terminal nucleotide of this oligonucleotide terminal region anneals to the first junctional nucleotide of the probe's first oligo positioning region. 

A first ligatable free end is then created at the nucleotide of each of the plurality of templates that is annealed to the junctional nucleotide of its respective probe's first template complementarity region. As in the uniplex embodiments, the ligatable free end may readily be created using exonucleases; in embodiments in which template complementarity to probe is continuous, ligatable free ends may also readily be created using single strand-specific endonucleases. 

In some embodiments, the sequence of the first oligonucleotide positioning region is identical among all of the plurality of probes. In such embodiments, a single species of first oligonucleotide may be used, thus appending identical oligonucleotides, and thus identical priming sites, to each of the plurality of templates. 

In other embodiments, the sequence of the first oligonucleotide positioning region differs among the plurality of probes, and a different species of first oligonucleotide is appended to each of the plurality of templates of distinct sequence. Even in such embodiments, however, the plurality of first oligonucleotides may include a priming site that is identical thereamong. The priming site in such cases is typically positioned in optional oligonucleotide region 24. 

As in the uniplex embodiments of the methods of the present invention, the multiplex embodiments may be iterated to attach additional oligonucleotides to each of a plurality of templates of distinct sequence in one or more additional reactions. 

In alternative embodiments, each of the plurality of probes further includes a second oligo positioning region directly adjacent to its template complementarity region. Template, respective first oligonucleotide, and respective second oligonucleotide are concurrently annealed to a respective one of a plurality of probes, followed by creation of first and second ligatable free ends and subsequent ligation. 

Each first oligonucleotide may usefully include a first priming site that is common thereamong, and each second oligonucleotide may usefully include a second priming site that is common thereamong. In such embodiments, subsequent amplification of each of the templates of distinct sequence may be effected using common first and second primers. When the first and second priming sequences are reverse complements of one another, bidirectional amplification may be effected using a single common primer. 

The multiplex methods of the present invention may include the further steps of separating the plurality of templates from the plurality of probes and oligonucleotides, and then amplifying the plurality of templates in a common reaction. 

In multiplex embodiments of the methods of the present invention, the first and/or second oligonucleotide may usefully include a genotypic label ("bar code tag") that permits the separate identification of each unique template or product amplified therefrom. 

Bar code tags are short nucleic acids having sequence that is designed algorithmically to maximize discrimination on a microarray displaying complements of the respective tags; a 1:1 correspondence as between tag sequence and nucleic acid to which it is appended permits each such nucleic acid to be identified by detection of the bar code uniquely associated therewith. See, e.g., Shoemaker et al., Nature Genet. 14(4):450-6 (1996); EP 0799897; Fan et al., Genome Res. 10:853-60 (2000); and U.S. Pat. No. 6,150,516, the disclosures of which are incorporated herein by reference in their entireties. 

In the methods of the present invention, a distinct bar code sequence may be included in each species of first and/or each species of second oligonucleotide. In these embodiments, the terminal region of each species of oligonucleotide is distinct in sequence, and can anneal only to a single species of probe. The 1:1 correspondence as between tag sequence and template-appended oligonucleotide thus permits each template or product amplified therefrom to be identified by detection of the bar code uniquely associated therewith. 

In other embodiments, bar codes may be appended to template 10 independently from oligonucleotides 20. 

FIG. 5 (see Original Patent) schematizes embodiments of the present invention in which a plurality of oligonucleotides are appended in series to a single ligatable free end of template 10. 

In these embodiments, probe 50 first oligo positioning region, referred to as 30 in previous figures, comprises at least subregions 34 and 32: oligo positioning subregion 32 is complementary to oligonucleotide 20 terminal region 22; subregion 34 is complementary to oligonucleotide 21. 

In the first step of the method, template 10 and at least oligonucleotide 21 are annealed concurrently to probe 50. Typically, oligonucleotide 20 is also concurrently annealed to the probe. If not present, a ligatable free end is created on template 10, and template 10 is then ligated to oligonucleotide 21. Oligonucleotide 20 is ligated in series to oligonucleotide 21, either in a separate reaction or concurrently with ligation of oligonucleotide 21 to template 10. 

Although shown as appended to a free 3' end of template 10, oligonucleotides 21 and 20 may be appended to a free 5' end of template 10. 

In these embodiments, oligonucleotide 21 may include a bar code, thus permitting the bar code to be appended to the template independently of oligonucleotide 20, which optionally (but typically), includes a priming site. 

When bar codes are appended to each of a 5' and 3' free end of template 10, the two bar codes uniquely associated with each template can be reverse complements of one another or different in sequence from one another. 

Appending common first and second priming sites directly to each of the plurality of templates of distinct sequence--without prior amplification of the template--facilitates the subsequent stoichiometric amplification of a wide variety of templates of distinct sequence, obviating the problems of unequal amplification observed with many multiplex PCR approaches. By permitting the de novo design of the priming sites, independently of considerations of template sequence, the methods of the present invention also permit amplification with primers having optimal hybridization characteristics, decreasing artifacts such as primer dimer formation.
 

Claim 1 of 20 Claims

1. A method of appending a first oligonucleotide and a second oligonucleotide directly to a nucleic acid template, the method comprising: annealing the template, the first oligonucleotide and the second oligonucleotide to a probe, wherein said probe includes at least a first template complementarity region and at least a first oligo positioning region directly adjacent thereto, the nucleotide of the first template complementarity region and the nucleotide of the first oligo positioning region that are directly adjacent within said probe being first junctional nucleotides that define a first probe junction therebetween; wherein said probe further includes a second oligo positioning region directly adjacent to said first template complementarity region, the nucleotide of the first template complementarity region and the nucleotide of the second oligo positioning region that are directly adjacent within said probe being second junctional nucleotides that define a second probe junction therebetween; wherein said first oligonucleotide includes a terminal region that is complementary to the first oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the probe's first oligo positioning region; and wherein said second oligonucleotide includes a terminal region that is complementary to the second oligo positioning region of said probe, the terminal nucleotide of said terminal oligonucleotide region being annealed to the junctional nucleotide of the second oligo positioning region; creating a first ligatable free end at the template nucleotide that is annealed to the junctional nucleotide of the probe's first template complementarity region; and then ligating said first oligonucleotide to said first free ligatable end to append said first oligonucleotide to the nucleic acid template; and creating a second ligatable free end at the template nucleotide that is annealed to the second junctional nucleotide of the first template complementarity region; and ligating said second oligonucleotide to said second ligatable free end; and then separating said template from said probe and said oligonucleotides. 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.