WO2010048386A1

WO2010048386A1 - Methods of sample preparation for nucleic acid analysis for nucleic acids available in limited amounts

Info

Publication number: WO2010048386A1
Application number: PCT/US2009/061655
Authority: WO
Inventors: William Pierceall; Kathleen Steinmann; Marie Causey; Tal Raz; Mirna Jarosz; Philip Buzby; John Thompson
Original assignee: Helicos Biosciences Corporation
Priority date: 2008-10-24
Filing date: 2009-10-22
Publication date: 2010-04-29

Abstract

The disclosure provides methods for preparation of nucleic acid samples available in limited amounts for nucleic acid analysis, such as, e.g., single molecule sequencing. The methods involve uses of carrier compositions that reduce loss of the target nucleic acid during further sample manipulation.

Description

METHODS OF SAMPLE PREPARATION FOR NUCLEIC ACID ANALYSIS FOR NUCLEIC ACIDS AVAILABLE IN LIMITED AMOUNTS

Related Application

This PCT application claims the benefit of and priority to U.S. provisional application serial number 61/108,382, filed October 24, 2008, the contents of which are incorporated by reference herein in their entirety

Technical Field

The invention is in the field of molecular biology and relates to methods for nucleic acid analysis. In particular, this invention relates to methods for preparation of nucleic acid samples available in limited amounts for nucleic acid analysis, such as, e.g., single molecule sequencing.

Background

Current nucleic acid and transcript profiling analysis methods involve cumbersome sample preparation and are susceptible to sample bias. Additionally, many biological samples, e.g., tissues or cells from human individuals, are available only in very limited quantities. For example, to overcome the sample limitation issues most sample preparation methods include the use of one or more methods of nucleic acid amplification. These methods often introduce amplification biases and/or sequence variations from the original sample that will affect the accuracy of the resulting sequence analysis.

Recently methods for high throughput, next-generation nucleic acid analysis involving sequencing have been demonstrated, some of which are able to visualize individual single molecules. Some of these methods involve a first and/or second amplification in the process, e.g., emulsion PCR followed by amplification of surface bound molecules to produce a clonal colony. In some methods prior to the amplification, steps which ligate on appropriate adapter(s) are also performed. Each of theses steps introduces biases, complexity, and costs. The products of these amplifications become the nucleic acid material that is actually sequenced and analyzed which may or may not accurately reflect the nucleic acid originally isolated from the sample.

An example of asynchronous single molecule sequencing-by-synthesis is illustrated in Figure 1. As shown, oligonucleotides 30-50 bases in length are covalently anchored at the 5' end to glass cover slips. These anchored strands perform two functions. First, they act as capture sites for the target template strands, if the templates are configured with capture tails complementary to the surface bound oligonucleotides. They also act as primers for the template- directed primer extension that forms the basis of the sequence reading. The capture primers are a fixed position site for sequence determination. Each cycle consists of adding the polymerase- labeled nucleotide analog mixture, rinsing, optically imaging the field containing millions of active primer template duplexes, and chemically cleaving the dye-linker to remove the dye. The labeled nucleotides are added either individually in a cycle or if the detectable moiety is spectrally resolvable more than one nucleotide can be added per cycle. The nucleotide analogs are such that they add only once per strand/cycle, e.g., a reversible terminator. The cycle (synthesis, detection, and dye removal) is repeated up to 25, 50, 100 times and, possibly, more.

The real-time single molecule sequencing-by-synthesis technologies rely on the detection of fluorescence from nucleotides as they are incorporated into a nascent strand of DNA that is complementary to the template being sequenced. The fluorophore may be released from the nucleotide by the polymerase during the catalysis step which grows the primer by nucleotide addition. This type of detection depends upon the ability of the imaging system to differentiate which of the four spectrally resolvable fluorescent nucleotides in the polymerase-labeled nucleotide mixture incorporates as the polymerase copies the template in near real time.

Another example of sequencing utilizes a ligase and labeled synthetic oligonucleotides rather than a polymerase and labeled nucleotides. A sequencing cycle is performed by ligase- mediated interrogation of one or more bases located 2-8 bases away from the site of ligation. Repeating cycles of ligation, imaging, cleavage of the oligonucleotide to remove dye and creating a new interrogation site, are performed until a sequence is determined.

Four major high-throughput sequencing platforms are currently available: the Genome Sequencers from Roche/454 Life Sciences (Margulies et al. (2005) Nature, 437:376-380; U.S. Patents Nos. 6,274,320; 6,258,568; 6,210,891), the IG Analyzer from Illumina/Solexa (Bennett et al. (2005) Pharmacogenomics, 6:373-382), the SOIiD system from Applied Biosystems (solid.appliedbiosystems.com), and the Heliscope™ Sequencer from Helicos Biosciences (see, e.g., U.S. Patent App. Pub. No. 2007/0070349 and the illustration in Figure 1). Although these new technologies are significantly cheaper compared to the traditional methods, such as gel/capillary Gilbert- Sanger sequencing, the sequence reads produced by the new technologies are generally much shorter (-25-40 vs. -500-700 bases). For example, the average read lengths on the four major platforms are currently as follows: Roche/454, 250 bases (depending on the organism); Illumina/Solexa, 25 bases; SOIiD, 35 bases; HeliScope, 25 bases.

There is a need for methods that overcome the issues of sample preparation associated with having limited amounts of sample.

Summary

The invention provides methods for nucleic acid preparation from samples in which target nucleic acids are available in limited quantities. The methods permit direct analysis of such nucleic acids without amplification. In preferred applications of the methods, the analysis performed involves single molecule sequencing. Methods of the invention may reduce sample bias, provide improved transcript counting, and information content.

In general, methods of the invention include: a) isolating a nucleic acid; b) optionally, fragmenting the nucleic acid; c) adding a carrier; d) modifying both nucleic acid and carrier; e) separating the modified carrier from modified nucleic acid; f) optionally, treating the modified nucleic acid a second time to remove or degrade residual carrier; g) anchoring directly or indirectly the modified nucleic acid to a support; and h) sequencing at least a portion of the nucleic acid

In more specific embodiments, the methods include: a) providing a sample, comprising a low abundance target nucleic acid (e.g., lower than 100, 50, 10, or 5 copies per genomic equivalent (~3 pg for human genome)); b) combining said sample with a carrier composition that reduces losses of the target nucleic acid during further sample manipulation; c) modifying the target nucleic acid by adding thereto a universal binding tag (e.g., polyA-tailing); d) separating the carrier composition from the target nucleic acid; e) attaching the target nucleic acid to an analytical surface via the binding tag; and f) analyzing the attached target nucleic acid (e.g., by optically resolving individual target nucleic acid molecule directly or by observing resolvable tags associated with the target).

In certain embodiments, the carrier is synthetic oligonucleotide having more than one biotin attached. When the nucleic acid sample is DNA the oligonucleotide is also DNA and when the nucleic acid sample is RNA the oligonucleotide is also RNA. The biotins may be attached either to the 5 '-end, internally, or both using chemistry standard in the art. No biotins are attached on the 3'-OH and in one embodiment, not within 5-10 bases of the 3 '-end. The sequence of the oligonucleotide so labeled may be any sequence(s), however preferably a mixture especially with respect to the terminal 3 '-base, each of A, C, G, TAJ should be present. In certain embodiments, the carrier composition comprises a synthetic oligonucleotide (e.g., a uracyl-containing oligonucleotide). For example, the oligonucleotide may have one or more "U" substituted for "T" wherein one is near the 3 '-end of the carrier. In a preferred embodiment, the carrier is added so as to represent >90%, >99%, 99.9% or higher of the mass of the nucleic acid/carrier mixture.

In certain embodiments, the nucleic acid/carrier is modified enzymatically using terminal deoxynucleotidyl transferase (TdT) and dATP. Optionally, when the nucleic acid is RNA the enzyme polyA polymerase and ATP maybe be used to tail the RNA. Typically the reaction conditions will be such to add between 50-70 A's to the 3 '-end, however, in some cases the range may be anywhere between 10 — 100 A's.

In certain embodiments, biotin labeled carrier is removed using a support modified with streptavidin. The preferred supports are beads or magnetic beads. Beads are removed by filtration, centrifugation, or magnetic fields.

Additional methods and compositions of the invention are described in detail below.

Brief Description of the Drawings Figure 1 illustrates single molecule sequencing by synthesis Figure 2 provides an example of one type of chemistry/enzymology with the use of a modified carrier.

Detailed Description of the Invention

The invention provides methods for sample preparation for nucleic acid available in limited quantities, without the aid of any form of amplification, thereby enabling the direct analysis of nucleic acids found in the sample. Methods of the invention may reduce sample bias, provide improved transcript counting, and information content. Methods of the invention may provide the ability to count individual RNA (cDNA) molecules or gene copy number, to detect rare transcripts, to identify germ- line or somatic mutations (e.g., single nucleotide polymorphisms (SNPs), insertions, deletions, rearrangements), splice variants, and novel new genes/transcripts. The method of the invention enables within sample comparisons, e.g., gene sequences (regulatory elements, exonic, intronic), gene location, gene copy number, and gene expression levels, as well as comparisons between different samples.

Methods of the invention generally involve isolation of nucleic acid using methods standard in the art depending upon whether the target nucleic acid is DNA or RNA. The nucleic acid may be further processed to a defined average length, e.g., physical shearing or enzymatic digestion. The practice of the invention involves addition of a carrier which functions both biologically and chemically similar to the (sheared) nucleic acid from the sample. The carrier mass constitutes >50%, >75%, >90%, 99%, 99.9% or higher when compared to the mass of the nucleic acid from the sample.

In one embodiment, the carrier is comprised of nucleic acid. The carrier generally will be a synthetic oligonucleotide ranging from 10 to 100 bases in length. The carrier can be comprised of the same sequence or many different sequences which are either unique to the carrier or found in common with the nucleic acid sample. The carrier can also have one or more constituents such as PNA, LNA, or a peptide, as part of the composition. The carrier can also have a detectable label attached so as to assess removal efficiency.

In another embodiment, the carrier is modified so as to have attached an affinity tag. The affinity tag could be one member of a binding pair, e.g., biotin:streptavidin, hapten:antibody, or sugar: lectin. The binding pair may also be a nucleic acid sequence duplex, in which the sequence is unique to the carrier, e.g., so that one member binding pair is a complement of a sequence unique to the carrier nucleic acid. Additionally, the carrier may be labeled with more than one affinity tag or type of affinity tag.

In another embodiment, the carrier is modified with a unique reactive, structural feature which is not found in the sample nucleic acid. When the carrier is used in large excesses, e.g., more than IOOX — 100OX, over sample nucleic acid, it is preferable that all carrier molecules include at least one modification to effect its removal from the sample. For example, when the nucleic acid from the sample is DNA, the carrier may have the thymidine bases substituted with uracil bases. The substitution may be at any level desired from a single T base to 100% of the T's replaced with U's. In this embodiment, the carrier is treated with, for example, USER enzyme (New England Biolabs), which is a mixture of uracil DNA glycosylase and DNA glycosylase-Iyase Endonuclease VIII (New England Biolabs) to cleave the carrier at all dU incorporations.

In another embodiment, the carrier can be multiply modified to include more than one of the features described, e.g., modified to include affmity tag(s) and U reactive site(s). Preferably, the type and number of modifications should not change the desired reactivity of the carrier when compared to the sample nucleic acid so as to not introduce any biases.

In another embodiment, the sample nucleic acid/carrier mixture is subjected to an enzymatic or chemical process. The process modifies both the sample nucleic acid and carrier in a similar way. Exemplary methods utilize enzyme and dNTPs, ligase and short oligonucleotide adapters, or chemical agents such as ULS (www.kreatech.com/ ). Additionally, in some cases the mass of the carrier is dominant, the reaction conditions are optimized in such a way that reaction proceeds to similar extent whether the sample nucleic acid is present or not.

In another embodiment, the sample nucleic acid/carrier (comprised of nucleic acid) mix is subject to a 3 '-end tailing reaction. A polyA tail is generated on the free 3' OH of all sample nucleic acid/carrier fragments. The nucleic acid can be DNA or RNA. The tail may be enzymatically generated using terminal deoxynucleotide transferase (TdT) and dATP. Typically, a polyA tail containing 50 to 70 A nucleotides is used. The poly A tail facilitates hybridization of the nucleic acid to polyT primer molecules attached to a surface for sequencing as described below. In principle, polynucleotide tailing can be carried out with a variety of dNTPs (or heterogeneous combinations), e.g., dATP. dATP may be preferred because TdT adds dATP with predictable kinetics useful to synthesize a 50-70 nucleotide tail. Similarly, RNA may be labeled with polyA polymerase enzyme and ATP.

In another embodiment, the carrier is an oligonucleotide multiply labeled with biotin. The biotin is preferably not be located near the 3 '-end of the carrier. Following the polyA tailing, the mixture is incubated with a support having streptavidin (SA) attached. The preferred support is beads, including magnetic beads which can be removed by centrifugation or applying a magnetic field. Additionally, affinity column chromatography can be used. The carrier is bound to the beads via the biotin: SA interaction and the sample nucleic acid can be isolated essentially free of carrier. This process might need to be repeated more than once to remove substantially all the carrier. In some applications, the final sample nucleic acid to carrier mass ratio is >100, >10, >1 or approximately 1:1.

In another embodiment, the carrier is an oligonucleotide modified both with biotin(s) and U' s in the sequence. Following the polyA tailing, the mixture is incubated with SA coated beads. The beads containing the carrier are removed using centrifugation or magnet. Optionally, this process may be repeated 2 or more times. The isolated sample nucleic acid may still have an unacceptable level of carrier so follow up treatment is performed with the USER enzyme mixture to degrade any remaining carrier. The degraded carrier may be left in the final mixture.

In another embodiment, the carrier is an oligonucleotide modified with only U(s) in the sequence. Following the polyA tailing, the USER enzyme mixture is used to degrade the carrier. The degraded carrier may be left in the final mixture.

Samples for use in the invention may be obtained from whole organisms, cell lines, tissue, blood, bodily fluids, or any other biological source. Methods of the invention are especially useful in combination with single molecule sequencing techniques, such as are described in co-owned U.S. Patent No. 7,282,337, and co-owned U.S. patent application, serial number 11/496,275, each of which is incorporated by reference herein. Single molecule sequencing, which comprises sequencing individual strands of DNA or RNA on a surface such that each strand is individually optically resolvable, provides inexpensive, high-throughput, and accurate analysis of nucleic acids and preserves the digital nature of the sample.

Once sample is prepared, in a preferred embodiment, sequencing is conducted on a surface onto which primers are attached for sequencing-by-synthesis. In embodiments in which nucleic acid is polyA tailed, 'primers are oligo d(T) primers, which facilitate hybridization of the polyA tails to the primers. In a highly-preferred embodiment, polyA tailed nucleic acid (DNA/RNA) templates are hybridized to oligo d(T) primers and then "locked" into place. Locking is accomplished by the addition of dTTP until all A's on the polyadenylated tail of the template have a complement. However, because the A's and T's can slide relative to one another, in a second step, a limited number of dATP, dCTP, and dGTP are incorporated into the primer such that the primer and template are prevented from sliding (dissociating). For example, fill and lock can be performed in any of the following ways. In a first embodiment, dTTP and reversible terminator analogs of A, C, and G nucleotide are combined. In this method, the dTTP fill the complement to the poly- A sequence of the template, and the terminators lock the primer and template together such that they cannot slide relative to one another. In a second embodiment, dTTP is added and then washed away, followed by addition of the other 3 nucleotides. Finally, in a third embodiment, all 4 nucleotides are added sequentially starting with dTTP with washing steps following each nucleotide addition. dTTP and 1 nucleotide (e.g., dATP) are added and washed away, followed by dTTP and the next nucleotide (e.g., dCTP) and a wash, and finally the addition of dTTP with the last nucleotide (e.g., dGTP).

In another embodiment, nucleic acid strands prepared as described above are sequenced using single molecule sequencing. In single molecule sequencing, template/primer duplex are individually optically resolvable on a sequencing substrate. One version of single molecule sequencing is taught in co-owned U.S. Patent No. 7,169,560, and for example, in U.S. application, serial number 10/990,167, each of which is- incorporated by reference herein. Essentially, polyA DNA (RNA) is hybridized to poly dT primers attached covalently to an epoxide-coated glass surface as taught in co-owned U.S. patent application, serial number 61/034,141, incorporated by reference herein. Poly dT primed surfaces and their uses are disclosed in co-owned U.S. patent application, serial number 11/958,173, incorporated by reference herein. After rinsing, the surface-bound duplex is exposed to one or more dNTPs, or analogs, comprising a detectable label, and a polymerase enzyme under conditions sufficient for template-dependent sequencing-by-synthesis. In a preferred embodiment a single species of dNTP is added and in a highly-preferred embodiment, the dNTP is an analog comprising a detectable label and an inhibitor of subsequent nucleotide incorporation, both being attached to the dNTP by a cleavable linker. Upon incorporation, the analog prevents next base incorporation, thus yielding a single incorporation per reaction cycle (assuming the presence of a complementary nucleotide in the template). After a wash step, incorporated nucleotides are visualized and recorded by position on the surface. The linker is then cleaved and duplex are prepared for subsequent cycles of nucleotide addition. Upon completion of a user-determined number of addition cycles, each position on the surface (representing a single duplex) will have associated with it a number of nucleotides representing the sequence of additions (and hence the sequence of the template) at that duplex. Informatic methods, such as those taught in co-owned, U.S. patent application, serial number 11/347,350, incorporated by reference herein, are then used to compile the aligned sequence of the starting material.

In another embodiment, sequencing by synthesis is conducted with labeled nucleotides with 4 optically distinct dyes attached. The nucleic acid may be attached to a substrate so as to be individually optically resolvable, either as individual molecules or individual clusters of molecules. The cluster of molecules may be produced directly on the substrate from individual single molecules. The nucleotides may be modified in a way so as they are classified as reversible terminators, e.g., add once per addition cycle. The sequencing by synthesis process involves cycles of incubation of all 4 nucleotides with a polymerase, 4-color imaging, removing the label and terminator moiety, repeating cycles until desired read length obtained.

In another embodiment, sequencing by synthesis is conducted with labeled nucleotides with 4 optically distinct dyes attached. The nucleic acid may be attached to a substrate so as to be individually optically resolvable, either as individual molecules or individual clusters of molecules. The cluster of molecules may be produced directly on the substrate from individual single molecules. The nucleotides may be modified in a way so as they are not classified as (reversible) terminators, e.g., add many times per addition cycle. The sequencing by synthesis process involves the real-time monitoring of all 4 nucleotides incubated with a polymerase. In a specific embodiment, the label is removed during the addition to the 3 '-end of the primer so as to not inhibit further incorporations of labeled nucleotides.

Substrates for use in the invention can be two- or three- dimensional and can comprise a planar surface (e.g., a glass slide) or can be shaped. A substrate can include glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly( methymethacrylate)), acrylic copolymer, polyamide, silicon, metal (e.g., alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel), polyacrolein, or composites. Suitable three- dimensional substrates include, for example, spheres, microparticles, beads, membranes, slides, plates, micromachined chips, tubes (e.g., capillary tubes), micro wells, microfluidic devices, channels, filters, or any other structure suitable for anchoring a nucleic acid. Substrates can include planar arrays or matrices capable of having regions that include populations of template nucleic acids or primers. Examples include nucleoside-derivatized CPG and polystyrene slides; derivatized magnetic slides; polystyrene grafted with polyethylene glycol, and the like.

Substrates are preferably coated to allow optimum optical processing and nucleic acid attachment. Substrates for use in the invention can also be treated to reduce background. Exemplary coatings include epoxides, and derivatized epoxides (e.g., with a binding molecule, such as an oligonucleotide or streptavidin).

Various methods can be used to anchor or immobilize the nucleic acid molecule to the surface of the substrate. The immobilization can be achieved through direct or indirect bonding to the surface. The bonding can be by covalent linkage. See, Joos et al., Analytical Biochemistry 247:96-101, 1997; Oroskar et al., Clin. Chem. 42:1547-1555, 1996; and Khandjian, Mot. Bio. Rep. 11:107-115, 1986. A preferred attachment is direct amine bonding of a terminal nucleotide of the template or the 5' end of the primer to an epoxide integrated on the surface. The bonding also can be through non-covalent linkage. For example, biotin- streptavidin (Taylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991) and digoxigenin with anti- digoxigenin (Smith et al., Science 253:1122, 1992) are common tools for anchoring nucleic acids to surfaces and parallels. Alternatively, the attachment can be achieved by anchoring a hydrophobic chain into a lipid monolayer or bilayer. Other methods for known in the art for attaching nucleic acid molecules to substrates also can be used.

Finally, methods of the invention can be combined with statistical and informatic techniques, such as those disclosed in co-owned, U.S. patent application, serial number 61/034,138, incorporated by reference herein, in order to further increase the accuracy and reliability of results produced herein.

Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

EXAMPLES

Example 1

The following Example illustrates certain embodiments of the invention and does not limit the invention in any way.

First strand cDNA is made from S. cerevisiae mRNA via oligo-dT priming (Invitrogen Superscript 111 kit according to manufacturers instructions). The resulting cDNA is polyadenylated at its 3' end to yield approximately 50 dATPs. An aliquot of 20 ng of the cDNA sample is combined with KOAc (5OmM), tris base (2OmM), MgAc (1OmM) (for a final concentration of 10%), CoCI (25OpM), dATP (5OX the sample molarity), an Rl 10-labeled control oligonucleotide (32 bases in length with single biotin label and single U) as "carrier" used to assess the tailing efficiency (0.5 pmole). The reaction is denatured at 95°C for 5 minutes and quickly chilled on ice for an additional 2 minutes. 20 U of terminal transferase and ddTTP to terminate the strands are then added to the sample mix and incubated at 42°C for 1 hour followed by a 10 minute enzyme heat inactivation step (70°C).

Following ddTTP blocking, add 20 pi (equal volume to reaction mixture) of 5 mg/ml Dynal M-280 Streptavidin-coated beads (beads have been pre-washed in 2X Binding and wash buffer and resuspended in same to concentration indicated.) Mix and let stand at room temperature for 30 min. Mix by gentle agitation each 5 min during the 30 min binding step. Place tubes against magnet for 3 min. then transfer supernatant to new tube. To the recovered supernatant, add 20 μl deionized water and 20 μl of 5 mg/ml Streptavidin-coated beads, mix and let stand at room temperature for 30 min. Mix by gentle agitation every 10 min. Place tubes against magnet and let sit for 3 min. then transfer liquid volume to a tube containing 420 μl ddH₂O. Concentrate the polyA tailed nucleic acid (free from carrier) by transferring the entire volume onto a YMlO column (MilliPore) and spin at 1OK rcf for 25 min. Discard flow through. Wash the filter with an additional 480 μl deionized water and spin again for 25 min at 1OK rcf. Repeat filtrations. Invert column into a fresh tube and spin for 5K rcf for 30 sec. to recover polyA tailed nucleic acid. The material is then degraded with USER following supplier (NEB) instructions. The material is used directly without further purification.

The polyA cDNA is then hybridized to a surface comprising oligo dT primers (50-mers) as described in co-owned, U.S. Patent No. 7,282,337, incorporated by reference herein. Sequencing-by-synthesis is carried out for thirty 4 nucleotide addition cycles. The resulting sequence reads collected are then identified by alignment to the S. cerevisiae transcriptome reference. Each read is representative of a single molecule. The variation in cDNA length resulting from RNA degradation, or reverse transcriptase incomplete transcription, allowed for complete sequencing coverage of more highly expressed mRNAs. Approximately 1 million alignable reads are collected; allowing for expression detection approaching 10 tpm. Reads are aligned via a statistical counting method as described in co-pending US patent application, serial numbers 611021,465, and 61/034,138, using an error-tolerant read seeding method as described in co-pending, co-owned US patent application, 61/041,905, each of which is incorporated by reference herein. Figures 1 and 2 are referred to for certain details.

All publications, patents, patent applications, and biological sequences cited in this disclosure are incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method for determining the sequence of a nucleic acid, the method comprising: a) isolating a nucleic acid; b) optionally, fragmenting the nucleic acid; c) adding a carrier; d) modifying both nucleic acid and carrier; e) separating the modified carrier from modified nucleic acid; f) optionally, treating the modified nucleic acid a second time to remove or degrade residual carrier; g) directly or indirectly anchoring the modified nucleic acid to a support; and h) sequencing at least a portion of the nucleic acid.

2. The method of claim 1, wherein the modifying (step d) is catalyzed by an enzyme.

3. The method of claim 2, wherein the enzyme is terminal deoxynucleotidyl transferase.

4. The method of claim 1, wherein the carrier and the nucleic acid have similar reactivity.

5. The method of claim 4, wherein the carrier comprises a synthetic oligonucleotide, DNA or RNA.

6. The method of claim 5, wherein the carrier comprises one or more PNA or LNA moieties.

7. The method of claim 1, wherein the nucleic acid is a synthetic oligonucleotide, polynucleotide, DNA or RNA.

8. The method of claim 1, wherein the carrier comprises an affinity tag.

9. The method of claim 8, wherein the affinity tag is one member of a binding pair.

10. The method of claim 10, wherein the binding pair is comprised of biotin/streptavidin, hapten/antibody, or ligand/receptor.

11. The method of claim 8, wherein the carrier has a single affinity tag.

12. The method of claim 8, wherein the carrier has multiple affinity tags.

13. The method of claim 1, wherein the carrier removal is carried out using an affinity column, beads, surface, or magnet.

14. The method of claim 1, wherein the carrier is modified to contain a reactive site not present in the nucleic acid.

15. The method of claim 14, wherein uracil bases are substituted for thymidine bases when the nucleic acid is DNA.

16. The method of claim 15, wherein the carrier is degraded with USER.

17. The method of claim 1, wherein the carrier is modified to include both an affinity tag and a second reactive site.

18. The method of claim 1, wherein the degradation occurs following affinity removal of the carrier.

19. The method of claim 1, wherein the carrier is added so as to represent >90%, >99%, 99.9% or higher of the mass of the nucleic acid/carrier mixture.

20. A method of preparing a sample for nucleic acid analysis, the method comprising: a) providing a sample, comprising a low abundance target nucleic acid; b) combining said sample with a carrier composition that reduces losses of the target nucleic acid during further sample manipulation; c) modifying the target nucleic acid by adding thereto a universal binding tag; d) separating the carrier composition from the target nucleic acid; e) attaching the target nucleic acid to an analytical surface via the binding tag; and f) analyzing the attached target nucleic acid.

21. The method of claim 20, wherein the method is performed without, amplification of the target nucleic acid.

22. The method of claim 20, wherein step f) comprises optically resolving individual target nucleic acid molecule directly or by observing resolvable tags associated with the target.

23. The method of claim 20, wherein step f) comprises sequencing.

24. The method of claim 20, wherein the carrier composition comprises a synthetic oligonucleotide.

25. The method of claim 20, wherein step c) comprises poly A- tailing.

26. The method of claim 20, wherein the low abundance target nucleic represents less than 100 copies per genomic equivalent.

27. The method of claim 20, wherein the modification performed in step c) also modifiers the carrier composition.