JPH11151092A - Extension of dna and assay by using rolling primer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining a base sequence by providing a set of primers, forming a template containing a primer binding site, etc., amplifying and identifying a double stranded DNA in the extension of the primer, and shifting the primer binding site, etc. SOLUTION: This method for determining a base sequence comprises providing a set of primers each having an extension region containing a terminal nucleotide, a segment arranged on a template and a nucleotide having one or more less complexity, forming a template containing a primer binding site which is complementary to at least one of the primers of the above set and a polynucleotide, amplifying a double stranded DNA by extending the primer in the extending region for forming the double strand completely matching with the primer binding site of the template, identifying the nucleotide at the terminal of the primer by identifying the amplicon, shifting the binding site by one base by mutating the primer binding site of the template, and repeating these processes for determining the sequence of the polynucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates generally to DNA sequencing and analysis methods, and more particularly to a method for sequencing base by base by successive extension of oligonucleotide primers.

[0002]

BACKGROUND OF THE INVENTION Large-scale sequencing projects typically involve the production of a library of contiguous smaller clones of the portion of the polynucleotide whose sequence is to be determined. Genomic DNA is fragmented and inserted into a yeast artificial chromosome (YAC) or cosmid, which insert is then fragmented and inserted into a phage or plasmid vector for sequencing (eg, Hunk
apiller et al., Science, 254: 59-67 (1991)). Large-scale sequencing projects can be done with either a so-called "directed" or "random" strategy,
Both approaches involve at least one or two labor intensive steps in which the template is prepared for sequencing by one or another variation of the Sanger chain termination method.

[0003] Many proposals have been made to reduce or eliminate these labor intensive steps. For example, one directional strategy consists of the first round of sequencing using vector-specific "universal" primers, followed by repeated cycles of synthesis of new sequencing primers generated from the sequence information just obtained. Subsequent novel sequencing using new primers is included. In this manner, a newly determined sequence of primers can "walk" along a relatively large sequencing template without the need to fragment and subclone the template. The disadvantage of such an approach is that it is difficult to obtain a new primer at each cycle to perform the next round of extension. The process may become intolerably slow while waiting for the next primer to be synthesized, or for example, for 1 to 15 nucleotide long primers
Either it becomes impractical due to the need to maintain a library of primers of all possible sequences that can be more than × 10 ⁹ . Proposals have been made that require primers constructed from libraries of shorter oligonucleotides (eg, pentamers or hexamers) to alleviate this difficulty (eg, Kotl
Er et al., Proc. Natl. Acad. Sci., 90: 4241-4245 (1993); Ki.
eleczawa et al., Science, 258: 1787-1791 (1992)).
However, even a hexamer requires a library of at least 4096 oligonucleotides.

[0004] In addition to the problem of template preparation, as described above, both directional and random approaches have been
Use the Sanger chain termination method of sequencing which requires the generation of a set of DNA fragments, each fragment having a common origin and ending at a known base. The set of fragments is typically separated by high resolution gel electrophoresis. They must have the ability to distinguish very large fragments that differ in size by less than a single nucleotide. Unfortunately, some significant technical issues have led to the efficient use of the Sanger-based approach to use longer sequences or large-volume sequencing without significant capital and labor investment. Seriously hinders scale-up. Such problems include: i) the labor intensive gel electrophoresis separation process is difficult to automate and the data analysis requires excessive variability (eg, band broadening due to temperature effects, DNA sequencing). Introduces secondary structure compression in the fragment, heterogeneity in the separation gel, etc.); ii) its properties (eg, processivity,
Nucleic acid polymerases whose fidelity, polymerization rate, chain terminator incorporation rate, etc.) are often sequence-dependent; iii) bands typically spatially overlapping in the gel
detection and analysis of DNA sequencing fragments present in ol amounts; iv) low signal due to the distribution of the labeled moiety in hundreds of spatially separated bands without being concentrated to a single homogeneous phase; and v) For single lane fluorescence detection, the availability of dyes with appropriate emission and absorption properties, quantum yields, and spectral resolution. For example, Tr
ainor, Anal.Biochem., 62: 418-426 (1990); Connell
Et al., Biotechniques, 5: 342-348 (1987); Karger et al., Nuc.
leic Acids Research, 19: 4955-4962 (1991); Fung et al.,
U.S. Patent No. 4,855,225; and Nishikawa et al., Electrop.
horesis, 12: 623-631 (1991).

[0005] (i) it does not require high resolution electrophoretic separation of DNA fragments, (ii) reduces the number of templates required in large-scale sequencing projects, and (iii) reduces the number of target polynucleotides. Significant advances in sequencing technology can be achieved if alternative approaches that can accommodate simultaneous or parallel application become available for sequencing DNA.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel method and approach for determining the sequence of a polynucleotide.

It is another object of the present invention to provide a novel "primer walking" approach for sequencing that requires few primers to perform.

It is yet another object of the present invention to provide methods and kits for reducing the number of templates required for large-scale sequencing projects.

[0009] Another object of the present invention is to provide a method for rapidly analyzing the pattern of gene expression in normal and diseased tissues and cells.

It is a further object of the present invention to simultaneously analyze and / or sequence a population of thousands of different polynucleotides (eg, a sample of polynucleotides from a cDNA library or a sample of fragments from segments of genomic DNA). To provide methods, kits, and devices for

Yet another object of the present invention is to provide methods, kits, and devices for identifying a population of polynucleotides.

It is another object of the present invention to provide a method for sequencing a segment of DNA in a size range corresponding to a representative cosmid or YAC insert.

[0013]

SUMMARY OF THE INVENTION The present invention relates to a method for determining the nucleotide sequence of a polynucleotide. The method includes: (a) providing a set of primers, each primer of the set having a terminal nucleotide, a template-located segment, and an extension region containing one or more reduced complexity nucleotides. (B) forming a template containing the primer binding site and the polynucleotide, wherein the primer binding site is complementary to at least one primer of the set; Forming an amplicon from the template by amplifying double-stranded DNA selectively formed by extending primers from the set, forming a duplex perfectly matched to the site; (d) amplifier Terminal nucleotide of the extension region of the primer by identification of recon Identifying; (e) mutating the primer binding site of the template to shift the primer binding site by one nucleotide in the direction of extension; and (f) steps (c) to (c) until the nucleotide sequence of the polynucleotide is determined. A method comprising the step of repeating (e).

In the above method, the amplicon comprises:
It can be formed by the step of amplifying double-stranded DNA by polymerase chain reaction.

In the above method, the complexity reducing nucleotide is deoxyinosine, and the polymerase chain reaction and elongation to form double stranded DNA are performed by deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyinosine triphosphate. , And in the presence of thymidine triphosphate.

In the above method, the step of mutating the primer binding site of the template comprises:
The template placement segment is performed by extending and amplifying with primers containing nucleotides mismatched with adjacent nucleotides at the primer binding site of the double-stranded DNA, whereby the identity of the adjacent nucleotides is determined by oligonucleotide identification using the primers. It can be changed by direction mutagenesis.

The present invention also provides a method for determining the nucleotide sequence of a polynucleotide, comprising: (a) providing a first set of primers, wherein each first primer of the set is 3'- Having an extension region containing a terminal nucleotide, a template placement segment, and one or more complexity-reducing nucleotides; (b) containing a first primer binding site, a promoter, a polynucleotide, and a second primer binding site Providing a double-stranded DNA template, wherein the first primer binding site is capable of forming an extendable duplex with at least one first primer; (c) recognizing the promoter Double-stranded D using RNA polymerase
Producing a population of RNA transcripts from the NA template; (d) mutating a first primer binding site in the RNA transcript by extending a first primer that forms an extendable duplex therewith (E) removing the amplicon from the single-stranded DNA template, wherein the first primer binding site is shifted one nucleotide in the direction of extension and a single-stranded DNA template is formed; (F) identifying the 3 ′ terminal nucleotide of the first primer extended to form a single-stranded DNA template by identifying the amplicon; (g) determining the nucleotide sequence of the polynucleotide And (b) repeating steps (b) to (f) until completed.

In the above method, the step of producing a population of RNA transcripts may further include the step of removing DNA from the population.

In the above method, the amplicon comprises:
It can be formed by amplifying double-stranded DNA by the polymerase chain reaction.

In the above method, one or more of the reduced complexity nucleotides in the extension region of the first primer is 2'-deoxyinosine, 8-oxo-2'-deoxyadenosine, and 8-oxo-2'-deoxyinosine. It may be selected from the group consisting of guanosine.

In the above method, the step of removing DNA from the RNA transcript population may include a step of treating the population with DNase.

In the above method, the RNA polymerase may be T7 RNA polymerase.

The present invention also provides an oligonucleotide primer defined by the formula: 5'-X ₁ X ₂ ... X _k IRZNN X ₁ X ₂ ... X _k wherein each X i is i = 1, 2, ... k and Xi
Is 2'-deoxyadenosine, 2'-deoxycytosine, 2 '
An oligonucleotide as selected from the group consisting of -deoxyguanosine, thymidine, and 2'-deoxyinosine; I is 2'-deoxyinosine; R is diaminopurine, 2'-deoxyguanosine, and thymidine Z is selected from the group consisting of 8-oxo-2′-deoxyadenosine and 8-oxo-2′-deoxyguanosine; N is 2′-deoxyadenosine;
For oligonucleotide primers selected from the group consisting of 2'-deoxycytosine, 2'-deoxyguanosine, and thymidine; and k is in the range of 8-30.

In the above primer, X ₁ X ₂ ... X _k can be defined by the following formula: 5 ′-(G) _i I (GT) _j (T) _r (G) _m or 5 ′-(G ) _i I (TG) _j (T) _r (G) _m where: G is 2′-deoxyguanosine, T is thymidine, I is 2′-deoxyinosine, and i is 3
J is an integer between 3 and 5; r is an integer between 3 and 6; and m is an integer between 3 and 8.
Is an integer between

[0025]

The method of the present invention achieves these and other objects by selective extension along a template by template mutation and repetitive cycle nucleotide identification by primer advancement. An important feature of the present invention is the complexity reduction to reduce the number of primers required to anneal to all possible primer binding sites on the sequencing template.
y-reducing) nucleotides (referred to herein as "rolling primers"). Another important feature of the invention is the systematic replacement of at least one of the four nucleotides in the target polynucleotide with a cognate reduced complexity nucleotide or its complement. Sequencing is initiated by annealing a rolling primer that differs only in its terminal nucleotide to the primer binding site of the sequencing template, so that only the rolling primer whose terminal nucleotide forms a perfect complement to the template is the only extension product. Leads to the formation of After amplifying the double-stranded extension product to form an amplicon, the terminal nucleotide, and hence its complement in the template, is replaced by the identity of the amplicon (ide
ntity). For example, in a simple embodiment, terminal nucleotides can be identified by the presence or absence of amplicons in four vessels used for separate extension and amplification reactions. The primer binding site of the successfully amplified polynucleotide template is then mutated, for example, by oligonucleotide-directed mutagenesis, such that the subsequent rolling primer has one nucleotide compared to the binding site of the previous rolling primer. At the site that only shifts in the direction of extension to form a duplex that perfectly matches the mutant template. The steps of selective extension, amplification and identification are then repeated. In this manner, the primer "rolls" along the polynucleotide during the sequencing process, moving one base at a time along the template with each cycle.

Generally, this aspect of the invention is performed in the following steps: (a) providing a set of primers (ie, rolling primers), wherein each primer in the set comprises one or more Having an extended region comprising reduced complexity nucleotides and terminal nucleotides;
(b) forming a template containing the polynucleotide to be sequenced and the primer binding site,
Wherein the primer binding site is complementary to the extension region of at least one primer of the set; (c) annealing a primer from the set to the primer binding site, wherein the extension region of the primer is completely associated with the template. Form a matched duplex and double-stranded DN
Extending the primers to form A; (d) amplifying the double-stranded DNA to form an amplicon;
(e) identifying the terminal nucleotide of the extension region of the primer by the identity of the amplicon; (f) mutating the primer binding site of the template so that the primer binding site is shifted by one or more nucleotides in the direction of extension; Thereby efficiently shortening one or more nucleotide target polynucleotides; and (g) until the nucleotide sequence of the polynucleotide is determined;
a step of repeating the step (f).

An important feature of the present invention is that the method can be applied in parallel to many different polynucleotides by using oligonucleotide tags. In accordance with this aspect of the invention, each polynucleotide of the population is linked to an oligonucleotide tag for transferring sequence information to a tag complement on a spatially addressable array of such complements. . That is, a unique tag is linked to each polynucleotide of the population that can be copied and used to shuttle sequence information to its complement at a fixed location on the array of such complements. After the tag has hybridized to its complement, a signal is generated indicating the transferred sequence information. The sequence of the tagged polynucleotide is determined by repeated cycles of information transfer and signal detection at the position of the corresponding tag complement.

The use of tags that shuttle information to individual spatial locations, rather than sorting the entire population of target polynucleotides into such locations, provides at least two major advantages: Tags are much smaller molecular entities, so that the kinetics of diffusion and hybridization are much more advantageous. Secondly, tag loading at spatially discrete locations only needs to be sufficient for detection, while target polynucleotide loading needs to be sufficient for both biochemical processing and detection; Thus, far fewer tags need only be spatially loaded into individual sites.

An important aspect of this embodiment of the invention is to attach an oligonucleotide tag to each polynucleotide of the population such that substantially all different polynucleotides have different tags. As described more fully below, this is accomplished by taking a sample of a complete ensemble of tag-polynucleotide conjugates. Here, each tag has an equal probability of binding to any given polynucleotide.

The oligonucleotide tags used in the present invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having increased binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." The subunits of the tag complement may consist of monomers of the unnatural nucleotide analog, or they may include an oligomer or an analog thereof having a length in the range of 3-6 nucleotides, the oligomer being minimally cross-hybridized. Selected from the set to soy. In such a set, the duplex consisting of the complement of the oligomer of the set and any other oligomer of the set contains at least two mismatches. In other words, a minimally cross-hybridizing set of oligomers will form at most a duplex with at least two mismatches with the complement of any other oligomer of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and the length of the subunits, if the subunits are oligomers from a minimally cross-hybridizing set. In the latter case, in general, the number will be much less than the number of all possible sequences ^{where the} length of the tag is 4 ⁿ for a tag of n nucleotides in length. Preferred monomers for the tag complement include peptide nucleic acid monomers and nucleoside phosphoramidates having a 3′-NHP (= O) (O ⁻ ) O-5 ′ bond with adjacent nucleosides. The latter compound is referred to herein as N3'φP5 'phosphoramidate. Preferably, both the oligonucleotide tags and their tag complement comprise a plurality of subunits selected from a minimally cross-hybridizing set consisting of natural oligonucleotides 3-6 nucleotides in length.

Generally, this embodiment of the invention is practiced by the following steps: (a) attaching an oligonucleotide tag from the repertoire of tags to each polynucleotide of the population so that substantially all different polynucleotides are ,
Forming a tag-polynucleotide conjugate having different oligonucleotide tags attached thereto; (b)
Labeling each tag according to the identity of the terminal nucleotide of each polynucleotide selectively amplified with the rolling primer; (c) cleaving the tag from the tag polynucleotide conjugate; and (d) detecting Sorting the labeled tags onto a spatially controllable array of tag complements. Preferably, this process is repeated a number of times sufficient to uniquely identify each polynucleotide to be sequenced or to reconstruct a larger polynucleotide from randomly generated fragments.

In summary, the present invention provides a novel "primer walking" method for DNA sequencing.
In addition, the present invention is easily automated for parallel applications and procedures that require the production of large amounts of sequence information (eg, large-scale sequencing of genomic DNA fragments, mRNA
And / or cDNA fingerprinting, and high resolution measurement of gene expression patterns).

Definitions "Complement" or "tag complement" as used herein with respect to an oligonucleotide tag refers to the oligonucleotide tag that specifically hybridizes to form a perfectly matched duplex or triplex. Oligonucleotide. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double-stranded or single-stranded. Therefore,
When a triplex is formed, the term "complement" is meant to include either the double-stranded complement of a single-stranded oligonucleotide tag or the single-stranded complement of a double-stranded oligonucleotide tag. .

As used herein, the term “oligonucleotide” refers to the usual pattern of monomer-monomer interactions (eg, Watson-Crick)
Natural or modified monomers or conjugates that can specifically bind to a target polynucleotide by base pairing, base stacking, Hoogsteen or reverse Hoogsteen base pairing, etc.
(Including deoxyribonucleosides, ribonucleosides, their anomeric forms, peptide nucleic acids (PNA), etc.). Usually, the monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a small number of monomer units (eg, 3-4) to tens of monomer units. The oligonucleotide is a series of letters (e.g., "ATGCCTG")
Whenever indicated by, unless stated otherwise,
The nucleotides are from left to right in 5 'to 3' order, "A" indicates deoxyadenosine, "C" indicates deoxycytidine, "G" indicates deoxyguanosine, and "T" indicates thymidine. It is understood that Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilide, phosphoramidate, and the like. It will be apparent to those skilled in the art that when oligonucleotides having natural or unnatural nucleotides can be used, for example when enzymatic processing is required, an oligonucleotide consisting of naturally occurring nucleotides is usually required. .

An "extendable duplex" for primer annealing to a template is the 3 'of the primer in the duplex formed by such annealing.
The -terminal nucleotide and the second nucleotide from the 3'-end form a Watson-Crick base pair with adjacent nucleotides in the template, and the duplex allows the polymerase to extend the primer along the template. It means that it is stable enough. The term contemplates that there may be multiple mismatches in the duplex formed between the primer and the template.

An "exact match" for a duplex is
Means that the polynucleotide or oligonucleotide chains making up the duplex form a duplex structure with each other such that every nucleotide in each strand undergoes Watson-Crick base pairing with nucleotides in the other strand. I do. The term also includes the pairing of nucleoside analogs that can be used (eg, deoxyinosine, nucleosides with 2-aminopurine bases, etc.). With respect to triplex, the term consists of a perfectly matched duplex and third strand of a triplex, wherein all nucleotides are Hoogsteen or with the base pair of the perfectly matched duplex. Meaning to undergo a reverse Hoogsteen meeting.
Conversely, a "mismatch" between a tag and an oligonucleotide in a duplex is that a pair or triplet of nucleotides in a duplex or triplex is a Watson-Crick and / or Hoogsteen and / or reverse foo. It means that it cannot receive Gustyn's bond.

"Nucleoside" as used herein
And "nucleotide" include natural nucleosides and nucleotides, which include the 2'-deoxy and 2'-hydroxyl forms (e.g., Kornberg and Baker, DNA Repl.
ication, 2nd edition (as described in Freeman, San Francisco, 1992). As used herein, "natural nucleotide" refers to the four common natural deoxynucleotides A, C, G, and T. An "analog" with respect to a nucleoside is a modified base moiety and / or
Or a synthetic nucleoside having a modified sugar moiety (e.g., Scheit, Nucleotide Analogs (John Wiley, New Y
ork, 1980); Uhlman and Peyman, Chemical Reviews,
90: 543-584 (1990)). However,
They can hybridize specifically. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce probe complexity, increase specificity, and the like.

"Amplicon" as used herein
Means the product of the amplification reaction. That is,
A collection of identical, usually double-stranded, polynucleotides that are replicated from a few starting sequences. Preferably, the amplicon is produced by the polymerase chain reaction (PCR).

As used herein, "complexity reducing nucleotide" refers to (i) a cognate natural nucleotide when paired with any of more than one natural nucleotide, ie, the natural nucleotide that it replaces. Nucleotides) can form a stable duplex substantially equivalent to the same duplex containing (nucleotide), and (ii) can be processed by substantially the same enzyme as the cognate natural nucleotide. Refers to natural nucleotides. Preferably, the reduced complexity nucleotide does not show degeneracy or ambiguity when processed by a DNA polymerase. That is, if the reduced complexity nucleotide is present in the template being copied by the polymerase, the polymerase will incorporate a unique nucleotide at the site of the reduced complexity nucleotide. Similarly, if the reduced complexity nucleotide triphosphate is a substrate for a DNA polymerase, it will be incorporated at only a single nucleotide site (ie, one or the other, but not both, of its complement). Candidate complexity-reduced nucleotides are readily available in simple hybridization assays (eg, in melting temperature comparisons) and in incorporation assays where test polymerization is tested by conventional sequencing or incorporation of radiolabeled complexity-reduced nucleotides. Tested (see, eg, Bessman et al., Proc. Natl. Acad. Sci., 4
4: 633 (1958)). Preferably, "substantially equivalent stability" as used herein means that the melting temperature of the test 13-mer duplex is such that the melting temperature of the tested 13-mer duplex is determined by Kawase et al., Nucleic Acids Research, 14: 7727-.
As described in 7736 (1986), means within 20 percent of the melting temperature of the same duplex containing naturally occurring cognate nucleotides.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a "primer walking" approach to DNA sequencing. Here, a special set of primers is used for template copy and mutation. The number of different primers in this set is minimized by the use of reduced complexity nucleotide and primer combinations and the template mutation process. Within each cycle of copy and mutation, the nucleotides of the polynucleotide are identified and the sequencing template is shortened by one. Template shortening effectively results from mutations that convert nucleotides in the target sequence to nucleotides in the primer binding site.

In an important aspect, the present invention provides for the use of oligonucleotide tags that shuttle the sequence information obtained in a "bulk" or solution phase biochemical process to individual spatially controllable sites on a solid phase. Provide a method for sequencing a majority of polynucleotides in parallel. The signals generated at the spatially controllable sites convey the sequence information carried by the oligonucleotide tag. As explained more fully below,
Sequencing is preferably performed by alternating the cycle of identifying nucleotides by using rolling primers and the cycle of shortening the target polynucleotide.

In one aspect, the oligonucleotide tags of the invention comprise a plurality of "words" or subunits selected from a minimally cross-hybridizing set of subunits. Such a set of subunits cannot form a duplex or triplex with the complement of another subunit of the same set that has less than two mismatched nucleotides. Thus, the sequence of any two oligonucleotide tags in the repertoire forming a duplex is never "closer" than two nucleotides different
do not do. In certain embodiments, the sequence of any two oligonucleotide tags in the repertoire may be duplexed with, for example, the complement of another subunit of the same set whose subunits have less than three mismatched nucleotides. It can still be "further" separated, such as by designing a minimally cross-hybridizing set so that it cannot form. Usually, the oligonucleotide tags of the present invention and their complements are oligomers of natural nucleotides, so they can be conveniently processed by enzymes such as ligases, polymerases, nucleases, terminal transferases, and the like.

In another aspect of the invention, tag complements have been developed, typically for antisense therapeutics, having enhanced binding strength and increased specificity for polynucleotide targets. Consists of unnatural nucleotide monomers, including a series of compounds. As described above in the definition of "oligonucleotide", this compound can include a variety of different modifications of natural nucleotides (e.g.,
Modification of the base moiety, sugar moiety, and / or monomer-to-monomer linkage). Such compounds also
Includes oligonucleotide loops, oligonucleotide "clamps", and similar structures that promote enhanced binding and specificity.

Rolling Primer Preferably, the rolling primer is 15 to 30 nucleotides in length and has the following form: X ₁ X ₂ ... X _k YY... YN where X _i is a repeating subunit Y is a reduced complexity nucleotide or its complement; and N is any of A, C, G, or T, or any of the reduced complexity nucleotides such as deoxyinosine. Terminal nucleotide. A segment of X _i nucleotides (referred to herein as a “template-arranged segment”) is suitably arranged in a repeating subunit, such that the primer has a terminal nucleotide aligned with the first nucleotide of the target polynucleotide. And correctly registered in the primer binding site. Preferably, the recurring subunit has one primer
If it is out of registration by one or more repeating subunits, it is long enough so that it becomes too unstable to remain annealed to the template. Preferably, the repeating subunit is 4-8 nucleotides in length. As will become more apparent below, arranging the template-located segments as a series of identical subunits reduces the overall number of primers required in a set of rolling primers. Preferably, the template placement segment is selected from the group of two or more nucleotides, at least one of which is the complement of the reduced complexity nucleotide used. In a preferred embodiment, the underlined X _k is oligonucleotide-directed mutagenesis (eg, Current Protocols in Molecular B
Indicates the position at which the template is mutated by iology (a technique well described in John Wiley & Sons, New York, 1995).

The segments YY ... YN are referred to herein as:
It is called the "extension region" of the primer. This is because the primer is extended from this end along the template. Preferably, the extension is performed by a polymerase so that YY ... YN is in the 5 '→ 3' direction. However, this direction can be determined using other methods of elongation (eg,
By linking the oligonucleotide blocks as described in US Pat. No. 5,114,839, it may be 3 ′ → 5 ′. An important feature of the present invention is that extension occurs only when the terminal nucleotide N forms a Watson-Crick base pair with an adjacent nucleotide in the template. The extension region contains a minimum number of nucleotides greater than 2 that can form a stable duplex with the template, even if there is a mismatch at the X _k position. That is,
In a preferred embodiment, the duplex between the extension region and the template must be stable enough to perform oligonucleotide-directed mutagenesis. Preferably, the extension region contains 3 to 6 nucleotides, and most preferably 4 nucleotides. Preferably, Y is selected from the group consisting of deoxyadenosine (A) and deoxyinosine (I).

The number of rolling primers required for a particular embodiment depends on several factors, including the type of reduced complexity nucleotide used, the length of the primer, the length of the extension region, and the template. Contains the repeat subunit length of the placement segment. For example, the following set of primers (SEQ ID NOS: 1-6) has an 18 nucleotide long template-located segment consisting of 6 nucleotides of G and A subunits.

[0047]

[Table 1] When Y is A or I and N is A, C, I, or T, the above set of rolling primers includes 192 (= 6 × 2 ³ × 4) primers. Especially,
Each "YYY" represents all of the following sequences: AAA, AAI, AII,
AIA, IAI, IAA, IIA, and III. As can be seen from the above example, the template placement segment is available to shift the primer by one nucleotide in the direction of extension after any cycle. That is, if a primer from subgroup (5) is used in a cycle, the next primer to be used is selected from subgroup (6), and if a primer from subgroup (6) is used in a cycle, The next primer used is selected from subgroup (1) (and so on). If PCR is used to copy and amplify the template, the template is effectively shortened by one nucleotide each cycle.

[0048] Alternatively, the binding strength of the extended region is G at all positions except immediately adjacent to the terminal nucleotide.
By replacing A with I and diaminopurine (D) with A. That is, an alternative set of “YYY” sequences is DDA, DDI, DGI, DGA, GDI, GDA, GG
Including I, and GGA.

In another embodiment, the template-located segment comprises a reduced complexity analog for mutating the rolling primer binding site as sequencing proceeds, so that fewer such segments are required. You. For example, the following template placement segment can be used with an extension region that converts all template nucleotides to C.

[0050]

[Table 2] Primers p1 and p2 are used in an alternative manner.
Primers p1 and p2 both convert C to A at position 1 and C or A to C at position 3. This maintains two segments of very stable GC base pairs at either end of the primer when they anneal. Primer p2 contains additional deoxyinosine within the internal segment of the repeat GT dimer. Note that each repeat unit is exactly one nucleotide out of phase. Deoxyinosine at position 2 is
The primer binding site is converted to a primer binding site that forms a perfectly matched duplex with primer p1 with a shift of one nucleotide in the direction of extension. Therefore,
By alternating the use of primers p1 and p2,
The primer can be advanced one nucleotide in each cycle.

Sequencing with Rolling Primers Prior to sequencing, the target polynucleotide is processed so that one or more types of nucleotides are replaced by their cognate reduced complexity nucleotides. In a preferred embodiment, this is conveniently achieved by replicating the target nucleotide in a PCR in which dGTP is replaced by dITP. The template for sequencing is then prepared by attaching the target polynucleotide to the primer binding site. Typically, this is achieved by inserting the target polynucleotide into a vector that contains a primer binding site. Preferably, the primer binding site is in the 3 'direction with respect to the target polynucleotide so that primer extension can be performed with a DNA polymerase. Such insertions may be made using StuI or Ecl136I if the rolling primers described above are used.
Conveniently performed using a blunt-ended truncation restriction endonuclease such as I. These enzymes leave a three base sequence adjacent to the beginning of the target polynucleotide that is complementary to the primers described above. Preferably, a primer (referred to herein as a "T" primer) is located at the other end of the target polynucleotide such that it can be amplified by PCR. For example, sequencing can be initiated on such a template (SEQ ID NO: 9) in four separate reactions, shown below, assuming the use of the primers described above.

[0052]

[Table 3] Here "NNNN ... NNN" represents the target nucleotide and "BBBB ... BB" represents the complement of the T primer binding site for amplifying the sequence by PCR. The underlined sequence indicates the extended region of the rolling primer. The template placement segments of these primers were arbitrarily selected to correspond to the primers from subgroup (1) above. Assuming that the sequence of the polynucleotide adjacent to the rolling primer binding site is "TAIC" (to illustrate the method), only reaction 1 results in the formation of an amplicon, and the first nucleotide of the polynucleotide is Identified as T. Preferably, prior to amplification, the primer is, in a preferred embodiment,
Sequenase in the presence of dATP, dCTP, dITP, and dTTP
And extended with a high fidelity DNA polymerase such as For example, if a labeled primer is used and the extension product is separated from the non-extended primer, it should be understood that selective extension can also be performed in a single container. An important feature is that only primers whose terminal nucleotide forms the correct Watson-Crick base pair with the template are extended. Preferably, after extension, any single-stranded DNA in the reaction mixture is
Digested with a single-stranded nuclease such as g bean nuclease. After such extension and digestion, the remaining double-stranded DNA is then, in a preferred embodiment, again dAT
It is amplified in the presence of P, dCTP, dITP, and dTTP to generate an amplicon. Preferably, this amplification is between 5 and
Achieved by 10 cycles of PCR, resulting in very low or no chance of producing abnormal amplification products.

A sample of the amplicon from Reaction 1 was
Removed and split into four new containers containing the following primers from subgroup (2):

[0054]

[Table 4] Since the first nucleotide of the target polynucleotide was determined in a previous cycle, as shown, select a primer from subgroup (2) whose extended region has the form "IIAN". This is the underlined T of the lower chain.
, Which are mutated to C in any amplicon generated by oligonucleotide-directed mutagenesis. That is, the primer is an oligonucleotide that directs mutation at a site in the amplicon. Therefore, this “T” is converted to “C” in the amplicon. Since the second nucleotide of the target is A, reactions 7 and 8 both lead to the production of amplicons. Since only a single target polynucleotide is currently considered, any amplicon can be sampled for the next cycle. As described more fully below, if multiple polynucleotides are sequenced simultaneously, an additional "pooling" step must be performed.

As described above, one sample of the two amplicons contains primers from subgroup (3) having an extended region having the form "IAIN".
Dispensed into two new containers.

[0056]

[Table 5] Reactions 9 and 10 both produce amplicons; therefore, the third base is identified as "I". For the next cycle, this then leads to the selection of primers from subgroup (4) having an extension region having the form "AIAN", and the process is continued.

A significant increase in sequencing selectivity using RNA template selection can be achieved by extending the rolling primer with an RNA template and reverse transcriptase. Gaining selectivity, in part,
It results from the easy removal of unwanted DNA by nuclease digestion after the RNA template has been synthesized. The general scheme of this embodiment is illustrated in FIG. Double-stranded DNA (dsDNA) template to be sequenced (100)
Is linked between the RNA polymerase promoter and the rolling primer binding site, for example, by cloning into a suitable vector containing such elements. Using standard protocols, the vector is dsDN
A template (100) and linearized downstream of the rolling primer binding site, and the dsDNA template (1
The RNA copy of (00) and the binding site are synthesized using an RNA polymerase such as T7 RNA polymerase (110). After synthesis, the reaction mixture is treated with DNase to remove excess DNA and the RNA copy is purified.
To the purified RNA, an appropriate rolling primer (referred to herein as the "first primer") is added (130), and the rolling primer, which forms an extendable duplex with the RNA template, is reverse transcribed. It is elongated with an enzyme. After such extension, the RNA is hydrolyzed (eg, by heating and / or reverse transcriptase RNase).
SsD removed and obtained (by action by H activity)
NA (140) is amplified, preferably by PCR. Preferably, one of the primers in the PCR (referred to herein as a "second primer") comprises a promoter sequence for the next transcription; and more preferably, the other primer comprises a rolling primer binding Binds to the template placement segment of the site.

[0058] Rolling primers for this embodiment (i.e., the first primer) Preferred sets has the following _{form: X 1 X 2 ... X k} IRZNN wherein X ₁ X ₂ ... X _k Is a template-located segment as described above, I is deoxyinosine, R is selected from the group consisting of G and diaminopurine ("D"), and Z is 8-oxo-2-deoxyadenosine ("oxo -
A ") and 8-oxo-2-deoxyguanosine (" oxo
-G "), and N is selected from the group consisting of A, C, G, and T. In this embodiment, any nucleotides in the template are converted to either C or T by base pairing with Z in the extension and amplification steps. This is because whenever a primer with oxo-A at the Z position is selected, it can base pair with either G or T, but when used as a template, it has a T
This is because only the incorporation of is allowed. Similarly, whenever a primer with oxo-G at the Z position is selected, it can base pair with either A or C, but only allows incorporation of C when used as a template. R stands for "place saver," which simply provides a stable base pair with either T or C.
(diaminopurines are preferred over T due to the greater stability of TD base pairs).
Finally, I converts T to C. Obviously, G can also be used in this position. As described above, when template-placed segments of primers p1 and p2 are used, the total number of rolling primers required for sequencing is 128 (= 2 × 2 × 2 × 16).

Rolling primers of the above form can be used for automated DNA synthesis using conventional chemistry and phosphoramidite monomers for various nucleotide analogs, which are commercially available, for example, from Glen Research (Sterling, VA). It is easily synthesized on a machine.

Cross-hybridizer with minimal subunits
Construction of oligonucleotide tags from a growing set As noted above, an important embodiment of the present invention is described by Brenner in U.S. Patent Nos. 5,604,097; 5,635,400; and 5,654,413; / US9
No. 6,095,513, which includes the simultaneous sequencing of multiple target polynucleotides with oligonucleotide tags of the type disclosed herein, which are incorporated herein by reference.

The oligonucleotide tags and their complements used in the methods of the present invention can range in length from 12 to 60 nucleotides or base pairs; more preferably, they are 18 to 40 nucleotides or base pairs. Length range; and most preferably, they are 25-40
Range of nucleotides or base pairs in length. When constructed from antisense monomers, the oligonucleotide tags and their complements preferably range in length from 10 to 40 monomers, more preferably they are
It ranges in length from 12 to 30 monomers. Most preferably,
Oligonucleotide tags are single-stranded, and specific hybridization is a Watson-tag with tag complement.
Occurs through click base pairing.

After chemical synthesis, the library of tags is conveniently maintained as a PCR amplicon that contains primer binding regions for amplification and restriction endonuclease recognition sites to facilitate excision and binding to the polynucleotide. Preferably, the composition of the primers is selected such that the right and left primers have approximately the same melting and annealing temperatures. In some embodiments, either one or both primers and the tag may be used to allow the use of a “stripping” and exchange reaction that renders the construct containing the tag single-stranded at a selected region. Other flanking sequences consist of three or less of the four natural nucleotides. Such reactions are typically performed with a DNA polymerase (eg, T4
DNA polymerase), using the 3 'to 5' exonuclease activity of enzymes such as those described in Sambrook et al., Molecu
lar Cloning, 2nd edition (Cold Spring Harbor Laboratory,
New York, 1989).

As noted above, an important use of tags is to "shuttle" information from a target polynucleotide to a solid support containing the tag complement. Preferably, this step involves excising the tag-containing segment of the double-stranded template (eg, one or more restriction nucleases),
It is performed by separating it from the reaction mixture, denaturing and labeling the excised tag, and applying it to a solid support for detection.
This step can be performed in various ways using standard molecular biology techniques, one of which is exemplified below. Similarly, excised tags can be labeled in various ways, including radioactive, fluorescent, chromogenic,
Direct or indirect binding such as a chemiluminescent marker is included. Many comprehensive reviews of methodologies for labeling DNA and constructing DNA probes provide guidance applicable to labeling the tags of the present invention. Such reviews can be found in Kricka, Nonisotopic
DNA Probe Techniques (Academic Press, San Diego, 1
992); Haugland, Handbook of Fluorescent Probesand
Research Chemicals (Molecular Probes, Inc., Eugen
e, 1992); Keller and Manak, DNA Probes, 2nd edition (St.
ockton Press, New York, 1993); and Eckstein, Ed., Ol.
igonucleotides and Analogues: A Practical Approach
(IRL Press, Oxford, 1991); edited by Kessler, Nonradioacti
ve Labeling and Detection of Biomolecules (Springe
r-Verlag, Berlin, 1992);

Preferably, the tag comprises one or more fluorescent dyes (eg, Menchen et al., US Pat. No. 5,188,934; and B)
egot et al., disclosed in International Application No. PCT / US90 / 05565).

Solid support for tag complements Preferably, detection of sequence information occurs at spatially separated locations where the tags hybridize to their complements.
It is important that the detection of the signal from successive cycles of tag transfer relate to the same tag complement position throughout the sequencing operation. Otherwise, the sequence of the signal will not be an honest representative of the sequence of the polynucleotide corresponding to the tag and tag complement. This need is met by providing a spatially controllable array of tag complements. As used herein, "spatially addressable" means that the location of a particular tag complement can be recorded and tracked through sequencing operations. Knowledge of the identity of the tag complement is not critical; it is only important that its position be identifiable between cycles of tag transfer. Preferably, the regions containing the tag complement are separated (ie, do not overlap with regions containing different tag complements), resulting in more convenient signal detection. Generally, spatially controllable arrays are constructed by binding or synthesizing the tag complement on a solid support.

The solid supports for use in the present invention can have a wide variety of forms, including microparticles, beads, and membranes, slides, plates, micromachined chips.
d chip). Similarly, the solid support of the present invention comprises
It can include a wide range of compositions, including glass, plastic,
Silicone, alkanethiolate derivatized gold (alkaneth
iolate-derivatized gold), cellulose, low and high cross-linked polystyrene, silica gel, polyamide, etc. Preferably, a discrete population of particles is used such that each has a uniform coating (or population) of complementary sequences of the same tag (and none), or a single support or several supports. Either the body is used in spatially separated regions, each containing a uniform coating (or population) of complementary sequences to the same tag (and none). In the latter embodiment, the area of the region can vary according to the particular application, and typically the region is from a few μm ² (eg, 3-5) to a few hundred μm ²
(For example, 100 to 500).

The tag complement can be used with a solid support on which the tag complement is synthesized, or can be separately synthesized and attached to a solid support for use. For example, this is disclosed below: Lund et al.
Nucleic Acids Research, 16: 10861-10880 (1988); Albr
etsen et al., Anal. Biochem., 189: 40-50 (1990); Wolf et al., N.
ucleic Acids Research, 15: 2911-2926 (1987); or G
hosh et al., Nucleic Acids Research, 15: 5353-5372 (198
7). Preferably, the tag complement is synthesized on the same solid support and used with the same solid support, which may include various forms and may include various binding moieties. Such supports may include microparticles or arrays, or matrices, of regions where a homogeneous population of tag complement is synthesized. A wide range of particulate supports
It can be used in the present invention, it is a controlled pore glass (CPG),
Includes microparticles composed of highly crosslinked polystyrene, acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, etc., and are disclosed in the following exemplary references: Meth. Enzymol., Section A, 11-14.
7, 44 (Academic Press, New York, 1976); U.S. Patent Nos. 4,678,814; 4,413,070;
No. 046,720; and Pon, Chapter 19, Agrawal (eds.), Methods
in Molecular Biology, Volume 20 (Humana Press, Totow
a, NJ, 1993). Microparticle supports include commercially available nucleoside derivatized CPG and polystyrene beads (available, for example, from Applied Biosystems, Foster City, CA); derivatized magnetic beads; polystyrene grafted with polyethylene glycol (eg, TentaGel ^™ ,
Rapp Polymere, Tubingen Germany) and the like. The choice of the properties of the support (eg, material, porosity, size, shape, etc.) and the type of binding moiety used will depend on the conditions under which the tag is used. Exemplary binding moieties are described in Pon et al., Biotechniques, 6: 768-775 (1988);
ebb, U.S. Patent No. 4,659,774: Barany et al., International Patent Application No. PCT / US91 / 06103; Brown et al., J. Chem. Soc. Commun., 1
989: 891-893; Damha et al., Nucleic Acids Research, 18: 3.
813-3821 (1990); Beattie et al., Clinical Chemistry, 39:
719-722 (1993); Maskos and Southern, Nucleic Acids
Research, 20: 1679-1684 (1992). As described more fully below, when the tag complement is bound or synthesized on a microparticle, the population of microparticles is immobilized on a solid support to form a spatially controllable array.

As described above, tag complements can also be synthesized on a single (or several) solid support to form an array of regions uniformly coated with the tag complements. That is, within each region in such an array, the same tag complement is synthesized. Techniques for synthesizing such arrays are disclosed below: McGall et al., International Application No. PCT / US93 / 03767; Pease et al., Proc. Natl. Acad. Sci.
91: 5022-5026 (1994); Southern and Maskos, International Application No. PCT / GB89 / 01114; Maskos and Southern (cited above); Southern et al., Genomics, 13: 1008-1017 (1992); and Maskos and Southern. , Nucleic Acids Researc
h, 21: 4663-4669 (1993).

Preferably, the invention is practiced with microparticles or beads that are uniformly coated with the complement of the same tag sequence. Microparticulate supports and methods for covalently or non-covalently attaching oligonucleotides to their surface are well known and exemplified by the following references: Beaucage and Iyer (cited above); Gait (ed.) Ol.
igonucleotide Synthesis; A Practical Approach (IRL
Press, Oxford, 1984); and the above references. In general, the size and shape of the microparticles is not critical; however, since the use of minimal reagents and samples facilitates the construction and manipulation of many repertoires of oligonucleotide tags, it can be as small as a few μm (eg, 1-2 μm) in diameter. Several hundred μ
Fine particles having a size in the range of m (for example, 200 to 1000 μm) are preferred.

Preferably, a commercially available controlled pore glass (CP
G) or a polystyrene support is used as the solid support in the present invention. Such supports are available with base-labile linkers and the first nucleotide attached (eg, Applied Biosystems).
(Foster City, CA)). Preferably, microparticles having a pore size between 500 Angstroms and 1000 Angstroms are used.

In another preferred application, non-porous microparticles are used for their optical properties, which are advantageous when tracking large numbers of microparticles on a planar support (eg a microscope slide). Can be used. Particularly preferred non-porous microparticles are Bangs Laboratories (Carmel, IN)
Glycidal methacrylate (glycid) available from
al methacrylate) (GMA) beads. Such microparticles are useful in various sizes and are derivatized with various linking groups to synthesize a tag or tag complement. Preferably, a large amount of tagged microparticles (massiv
For the parallel operation in ely), GMA beads with a diameter of 5 μm are used.

Binding of Tags to Target Polynucleotides An important aspect of the present invention is to tag polynucleotides in a population (eg, a cDNA library) such that the same tag does not bind to different polynucleotides. This latter condition can be met essentially by linking the repertoire of tags to a population of polynucleotides, followed by cloning and sampling the linked sequence. The repertoire of oligonucleotide tags can be linked to a population of polynucleotides in a number of ways, such as, for example, direct enzymatic ligation, amplification using primers containing the tag sequence (eg, via PCR), and the like. The first ligation step results in a very large population of tag-polynucleotide conjugates, so that, in general, a single tag
Attached to many different polynucleotides. But,
By taking a sufficiently small sample of the conjugate,
The probability of obtaining “overlap”, ie the same tag on two different polynucleotides, can be negligibly small. (Note that it is also possible to obtain different tags with the same polynucleotide in the sample, which simply leads to the polynucleotide being processed twice (eg, sequenced). When multiple patterns of gene expression are analyzed, multiple tags with the same polynucleotide will be common occurrences and are expected due to differences in mRNA abundance). As described more fully below, the probability of obtaining an overlap in a sample can be estimated by Poisson distribution. This is because the number of conjugates in the sample is large (eg, on the order of thousands or more). Since the tag repertoire is large (for example, on the order of tens of thousands or more), the probability of selecting a specific tag is small. Preferably, the size of the tag repertoire is about 100 times the number of different species of polynucleotide in the population being analyzed. Alternatively, in other words, the complexity of the tag repertoire is preferably about 100 times that of the population of polynucleotides to be analyzed. In general, the larger the sample, the greater the probability of obtaining an overlap. Thus, the design trade-off is to choose a large sample tag-polynucleotide conjugate (which ensures sufficient coverage of the target polynucleotide in, for example, a shotgun sequencing operation) and to choose a small sample. (This ensures that there is a minimum number of overlaps). In most embodiments, the presence of duplication merely adds an additional source of noise or, in the case of sequencing, adds only a small complication to scanning and signal processing. This is because the region of the tag complement that provides a large number of signals simultaneously can be simply ignored. The term "substantially all" as used herein with respect to attaching a tag to a polynucleotide refers to the statistically independent sampling procedure used to obtain a population of tag-molecule conjugates. It means reflecting the nature.
With respect to the actual percentage of tag-molecule conjugate, virtually all meanings depend on how the tag is used. For nucleic acid sequencing, preferably substantially all means that at least 80% of the tags have a unique bound polynucleotide. More preferably, it means that at least 90% of the tags have a unique bound polynucleotide. Even more preferably, it means that at least 95% of the tags have a unique attached polynucleotide. And most preferably, it means that at least 99% of the tags have a unique attached polynucleotide.

In a preferred embodiment, the tag, the polynucleotide to be sequenced, the primer binding site,
And other elements for manipulating the sequence are inserted into the cloning vector to establish a basic library that can be sampled and amplified when needed. For example, such a construct may have the following form:

[0074]

[Table 6] Here, the "T" or tag binding site and the "S" or sequencing primer binding site, together with appropriate primers, are used to amplify the insert of the cloning vector to form a PCR amplicon for subsequent analysis. Is done. After the steps of PCR amplification and terminal nucleotide identification, the cleavage site is used to excise the tag from the amplicon. After amplification, as shown below
It is important that the target polynucleotide be protected from unwanted cleavage by the nucleases used in the identification step. Preferably, this is achieved by careful selection of methylation and restriction endonucleases.

A preferred embodiment for simultaneously sequencing targeting polynucleotides of a population of tagged polynucleotides is illustrated in FIG. 2a. Preferably, the population of tagged polynucleotides is dATP, dCTP,
Amplified from a vector as described above in the presence of dITP, and dTTP, a T primer binding site (12), a cleavage site (14)
(This is optional, as shown below), tags (1
6), a double-stranded DN containing a cleavage site (18), a target polynucleotide (20), and a rolling primer binding site (22)
This produces a population of A (10).

In the first population, the rolling primer binding site (22) contains a known complement to the extension region (24) (eg, AGG as shown in the Examples below). The initial population of samples is preferably transferred to four separate containers (28-34) (26). Here they are
Combined with the rolling primers of subgroup (1) above with the extension regions -AIIA, -AIIC, -AIIG, and -AIIT (four rolling primers are placed in one container and compete with each other for extension) However, if the primers are used separately, the error appears to be lower). Sub group
The rolling primers (1) to (6) are used herein to illustrate the invention. Obviously, many alternative forms of the rolling primer can be used. In subsequent cycles, the transfer step (26) becomes more complex, as described more fully below. This is because more than four vessels (ie, up to 32 (= 4 × 8) in the embodiments illustrated herein) are required for the extension reaction. After the double-stranded DNA (10) has been combined with a suitable rolling primer, the following step (36) is performed: the double-stranded DNA is denatured, for example, by heating; Lower temperature to anneal; dATP, dC
Primers are extended with a high fidelity DNA polymerase such as Sequenase in the presence of TP, dITP, and dTTP;
Preferably, any remaining single-stranded DNA, e.g., Mun
digested with a single-stranded nuclease such as g bean nuclease to reduce the potential for interference of residual single-stranded DNA in subsequent amplification; T-primers are added; and the double-stranded extension product is amplified, preferably Is amplified by 5 to 10 cycles of PCR to produce amplicon A (38), amplicon C (40), amplicon G (42), and amplicon T (44), respectively.

As an alternative to and / or as a supplement to the process of digestion with single-stranded nucleases, double-stranded DNA (10)
Can be treated with methylase (or equivalently, amplified in the presence of 5-methylcytosine triphosphate). After such treatment, any double-stranded DNA that is not the product of at least two extension reactions is hemimethylated or fully methylated at the cleavage site (18). Thus, nucleases recognizing site (18) on their sequence will not cleave it.
If a sample of the amplicon is obtained by a capture agent such as biotin on the T primer, cleavage of the cleavage site (18) with a nuclease can release the tag for analysis. However, sites that are methylated or hemimethylated are not cleaved to produce spurious signals on application of the tag to the solid support (48).

After a sample has been obtained from each amplicon, the tag is excised by cleavage sites (14) and / or (18) and labeled (46), as described more fully below. The labeled tags are then individually applied to their tag complement on a solid support (48) or pooled, depending on factors such as the labeling system used, the complexity of the tag mixture, And either applied to the support. A sample of the amplicon is also obtained for further processing (50-56) according to the method of the invention. Depending on the identity of the most recently determined nucleotides and the identity of the current extension region, samples are individually dispensed into containers containing rolling primers for the next cycle, or It can be combined with one or more other samples and dispensed into containers containing rolling primer for the next cycle. Unlike the case with one polynucleotide, when a population of polynucleotides is sequenced, all containers almost always contain amplicons as the conclusion of an amplification reaction. Thus, after extension, digestion, and amplification,
The amplicons in containers 28, 30, 32, and 34 each have a T, G at the starting position (or more generally, the nucleotide position adjacent to the rolling primer binding site).
(Or I), corresponding to a target polynucleotide having C, and A. With this information and knowledge of the sequence of the extended region of the current amplicon, the next cycle of rolling primers can be selected. As with one polynucleotide, in each successive cycle, the rolling primer is selected such that it shifts or advances the rolling primer binding site by one or more nucleotides along the template in the direction of rolling primer extension. . Preferably, one nucleotide shift occurs in each cycle. As described above, the rolling primer selected for the extension step also serves to generate mutations in the template during amplification. This mutation changes most nucleotides inside the extension region to nucleotides that are complementary to the templated segment of the rolling primer in the current cycle. In the following table, the primer selection and amplicon pool patterns in cycles 2-4 of the sequencing operation are described with respect to the above embodiments. In the first cycle, the first template is dispensed into four containers for denaturation and extension.

[0079]

[Table 7] The nucleotide to the right of the line between the nucleotides in the second column is the terminal nucleotide of the rolling primer used to produce the amplicon. In general, the algorithm for determining the next cycle of the rolling primer is as follows: (i) the end of the terminal nucleotide of the extension region of the current rolling primer (leftmost of the "IIA" sequence in the second column) (Ii) determine whether nucleotide I or A is complementary to the nucleotide that base-paired with the terminal nucleotide (ie, in the above example: amplicon A “A” for amplicon C, “A” for amplicon C (because A bases with I and C), and “I” for amplicon G (because I bases with C) ), And "I" for amplicon T (because I also forms a base with A)), (iii) determining the determined nucleotide I or A to the left of the terminal nucleotide Insert For this embodiment,
The general pattern of change between extended region sequences is illustrated in FIG. 2b. Longer stretched regions lead to more complex patterns, but the basic algorithm that defines acceptable changes remains the same.

[0080]

[Table 8]

[0081]

[Table 9] Typically, the 8th cycle requires 32 reactants and continues to be required in each cycle until sequencing stops.

Clearly, the further steps outlined above are performed, for example, to separate the initial extension products from unsuitable single-stranded DNA and / or single-stranded nucleases (if used). You. Operations such as polynucleotide and other reagents, temperature control for PCR,
Commercial laboratory robots (eg, Biomek 1000 (Beckman
Instruments, Fullerton, CA)).

The rolling and T primers are described in Ji et al., Anal. Chem. 65: 1323-1328 (1993); Cantor
Et al., As taught by US Pat. No. 5,482,836, etc., can be constructed to have double-stranded segments capable of binding to an anchored single-stranded oligonucleotide by triplex formation for separation. Thus, for example, magnetic beads carrying such single-stranded oligonucleotides can be used to capture amplicons and, for example, selectively amplified double-stranded DNA (other DNA is not amplified and therefore Can be used to transfer them to individual containers containing nucleases that cleave the tags at the cleavage site 18 (which remains hemimethylated). Preferably, the T primer contains 5 'biotin which allows the released tag to be captured and conveniently labeled.
For example, after capture by avidinated magnetic beads, the 3 'strand of the double-stranded segment is exposed to an enzyme such as T4 DNA polymerase in the presence of deoxynucleoside triphosphate (dNTP) corresponding to the nucleotide adjacent to the tag. The tag is removed by use. Thus, if there are no other adjacent nucleotides along the strand to the 3 ′ end, the 3 ′ → 5 ′ exonuclease activity of the polymerase will remove the 3 ′ strand back to the adjacent nucleotide, at which point the adjacent An exchange reaction is initiated to prevent further stripping past the nucleotides. The 3 'end of the tag can then be labeled with a labeled dNTP in an extension reaction. After labeling, the non-biotinylated strands are removed by denaturation, and after labeling tags have been hybridized to their tag complement and detected, which can be applied to a spatially controllable array for detection, The tags are removed by washing and a label tag from the next set of amplicons can be applied.

Detection signal at spatially controllable site
Preferably a device for measuring the spatially controllable array is established by fixing the fine particles containing tag complements to a solid surface. Whenever a light production signal (eg, chemiluminescence, fluorescence, etc.) is used, various devices can be used to detect hybridized tags and / or enzymatic events on such arrays. . For example, scanning systems may be used, such as those described in International Patent Applications PCT / US91 / 09217, PCT / NL90 / 00081, and PCT / US95 / 01886. Preferably, the microparticles containing the tag complement are loaded into the flow chamber as a liquid particle slurry. So, they are the DNA on the microparticles on the substrate
Is held in place by a combination of non-specific binding of the GPC and gentle flow pushing the particles toward the flow chamber barrier. An exemplary apparatus is illustrated in FIG. 3: the flow chamber (500) uses standard micromatching techniques (eg, Ekstrom et al., International Patent Application PCT / SE91 / 00327;
rown, U.S. Patent No. 4,911,782; Harrison et al., Anal.
m. 64: 1926-1932 (1992)) by etching a cavity having a liquid inlet (502) and an outlet (504) in a glass plate (506). The dimensions of the flow chamber (500) are
(Eg, GMA beads) are sized so that they can be placed in a tightly packed planar monolayer cavity (510) of 100,000 to 200,000 beads. The cavity (510) is created in a closed chamber with entrance and exit by anodic bonding of a glass coverslip (512) on an etched glass plate (506) (see, eg, Pomerantz, US Pat.
3,397,279). With the glass coverslip in place, the cavity (510) has a height several tens percent greater than the diameter of the microparticle to confirm that a monolayer is formed. A barrier or shelf adjacent to the outlet (504) is present in the glass plate (506), which forms a barrier to the particulates of the slurry, but at the same time allows the liquid components of the slurry or other reagents to pass freely. I do. Reagents are regulated (514 to 520) from the syringe pump into the flow chamber through a valve block (522) controlled by a microprocessor, as commonly used in automated DNA and peptide synthesizers (eg, Bridgh
Am et al., U.S. Pat. No. 4,668,479; Hood et al., U.S. Pat.
52,769; Barstow et al., US Patent No. 5,203,368; Hunkapil.
ler, U.S. Pat. No. 4,703,913; etc.).

In particular, the hybridized tag is detected by exciting its fluorescent label with an illuminating light beam (524) from a light source (526), which may be a laser, mercury arc lamp, etc. . The illuminating light (524) passes through the filter (528) and excites the fluorescent label on the tag complement that specifically hybridized to the tag complement in the flow chamber (500). The resulting fluorescence (530) is collected by a confocal microscope (532), passed through a filter (534), and directed to a CCD camera (536). This creates an electronic image of the bead array for processing and analysis by the workstation (538). Preferably, about 25 n
The M concentration of the tag is 50 mM NaCl, 3 mM Mg, 10 mM Tris-HCl (pH
Is passed through the flow chamber in a hybridization buffer consisting of 8.5), after which the fluorescent label carried by the tag is illuminated and the fluorescence is collected.
The tag is thawed from the tag complement by passing the hybridization buffer through the flow chamber at 55 ° C. for 10 minutes at a flow rate of 1-2 μL per minute.

In sequencing applications, microparticles can be immobilized on the surface of a substrate in a variety of ways. For fixation,
It should be strong enough to allow continuous cycles of reagent exposure and washing without significant loss. When the substrate is glass, its surface can be derivatized with an alkylamino linker using a commercially available reagent (eg, Pierce Chemical), which is then crosslinked to avidin, again using conventional chemistry. Can form an avidinated surface. Biotin moieties can be introduced into microparticles in a number of ways.

Kits for Performing the Methods of the Invention The present invention includes kits for performing the various embodiments of the present invention. Suitably, the kit of the invention comprises a set of rolling primers for performing extension and amplification according to the invention. The kit may also include a repertoire of tag complements attached to a solid support. In addition, kits of the invention may include a repertoire of corresponding tags (eg, as primers for amplifying the polynucleotides to be sorted or as elements of a cloning vector). Preferably, the repertoire of tag complements is bound to the microparticle. The kit also
Suitable buffers for enzymatic processing, detection chemistry (eg, fluorescent or chemiluminescent components for labeling tags, etc.), instructions for use, processing enzymes (eg, ligases, polymerases, transferases, etc.) may be included. . In important embodiments for sequencing, the kit may also include a substrate, such as an avidinized microscope slide or microtiter plate, for immobilizing the microparticles for processing.

[0088]

EXAMPLE 1 Construction of a Tag Library An exemplary tag library is constructed as follows to form a chemically synthesized 9-word tag of nucleotides A, G, and T defined by the following formula: Do:

[0089]

[Table 10] Here, “[ ⁴ (A, G, T) ₉ ]” indicates a tag mixture in which each tag consists of nine 4-merwords of A, G, and T; and “p” indicates 5 ′ phosphate . This mixture is ligated to the following right and left primer binding regions (SEQ ID NO: 10 and SEQ ID NO: 11):

[0090]

[Table 11] The right and left primer binding regions are ligated to the above tag mixture, then the single stranded portion of the ligated structure is filled with DNA polymerase, then mixed with the right and left primers shown below and amplified to form a tag library. Get.

[0091]

[Table 12] The underlined portion of the left primer binding region indicates the RsrII recognition site. The leftmost underlined region of the right primer binding region is Bsp1
20I, ApaI, and EcoO109I recognition sites, and HgaI
Indicates the cleavage site. The rightmost underlined region of the right primer binding region indicates the recognition site of HgaI. If necessary, the right or left primer can be used (conventional reagents, e.g., Clontech
Synthesized with bound biotin (using reagents available from Laboratories, Palo Alto, CA) and may facilitate amplification and / or purification after cleavage.

Example 2 Tag-Poly for cDNA "signature" Sequencing
Construction of Nucleotide Conjugate Plasmid Library pGGCCC anchored at the poly-A region border of mRNA
T ₁₅ and (A or G or C) as a primer for first strand synthesis, and using N ₈ (A or T) GATC as the primer for second strand synthesis by conventional protocol
Generated from mRNA samples. That is, both are degenerate primers, so that the second strand primer exists in two forms and the first strand primer exists in three forms. The GATC sequence in the second strand primer corresponds to the recognition site for MboI; other four base recognition sites can be used as well (eg, recognition sites for BamHI, SphI, EcoRI, etc.). Second
The presence of A and T adjacent to the recognition site of the strand primer ensures that stripping and exchange reactions can be used in the next step to generate a 5 base 5 ′ overhang of “GGCCC”. The first strand primer is annealed to the mRNA sample and extended with reverse transcriptase, after which the RNA strand is degraded by the RNase H activity of the reverse transcriptase, leaving a single-stranded cDNA. The second strand primer is annealed and extended with a DNA polymerase using conventional protocols. After second strand synthesis, the resulting cDNA was converted to CpG methylase (New England Biolabs, Be
verly, MA). The 3 ′ strand of the cDNA is then
Using T4 DNA polymerase in the presence of ATP and dTTP, the ends were deleted by the above-described removal and exchange reactions, and then the cDNA was ligated to the tag library of Example 1 previously cut with HgaI, and Get the construct of:

[0093]

[Table 13] Separately, the following cloning vector (SEQ ID NO: 12)
For example, Bluescript phagemid (Stratagene, La Jol
Starting from commercially available plasmids such as la, CA).

[0094]

[Table 14] Rolling primer binding sites are described in the subgroups above.
It corresponds to the rolling primer of (1). Plasmid P
Cleavage with pu MI and PmeI (to obtain RsrII compatible ends and blunt ends so that the insert is oriented)
Methylate with AM methylase. Tagged constructs with RsrII
And then ligated to the open plasmid, after which the conjugate is cut with MboI and BamHI to ligate and close the plasmid. The plasmid is then amplified and isolated for use as a template for extension and amplification according to the invention.

Example 3 Determination of Signature Sequence of cDNA Library The plasmid constructed in Example 2 was subjected to the above-mentioned rolling primer and the following T primer (SEQ ID NO: 1).
Used to generate extension products and amplicons, along with 3):

[0096]

[Table 15] Here, I is deoxyinosine added in order to balance the annealing temperature and the melting temperature of the T primer and the rolling primer. Preferably,
The annealing temperature is about 55 ° C. Obviously, numerous other arrangements can be used in the practice of the present invention. The above rolling primer is used.

A segment containing the rolling primer binding site from the T primer binding site is cut out from the plasmid of Example 2 and separated. (This can be done in various ways known to those skilled in the art, for example, by processing the plasmid to include restriction sites flanking the segment, or simply by PCR.
Can be achieved by direct amplification of After replacing deoxyguanosine with deoxyinosine (eg, by PCR in the presence of dITP), the segments are aliquoted into four containers, denatured, and the appropriate rolling primer is added. The conditions were adjusted to allow annealing of the rolling primer, and then the primer was used with a high fidelity polymerase such as Sequenase in the presence of dATP, dCTP, dITP, and dTTP using the manufacturer's protocol And elongate. Remaining single-stranded DNA
Is digested with a single-stranded nuclease (eg, Mung bean nuclease). Optionally, the double-stranded DNA extension product is reacted, for example, by capture via triplex formation (eg, between the T-primer binding region and the appropriate single-stranded complement attached to magnetic beads). It can be separated from the mixture.

Double-stranded DNA was combined with T primers (and rolling primers if a separation step was used) and amplified by 5-10 cycles of PCR in the presence of dATP, dCTP, dITP, and dTTP. To form the four first amplicons. The samples are combined and redistributed into containers with the appropriate rolling primer for the next cycle of extension. A sample is also withdrawn for analysis.

Preferably, the sample for analysis is S
The primers are separately captured on magnetic beads having a single-stranded sequence forming a triplex. The beads were then replaced with ApaI
(This cleaves the tag from the target polynucleotide)
Transfer to a reaction mixture containing The free chain containing the tag (SEQ ID NO: 14) is then captured via the biotinylated T primer by avidin-coated magnetic beads and transferred to a reaction vessel. Here, its 3 'end is removed in the presence of T4 DNA polymerase and dGTP as shown below:

[0100]

[Table 16] Here, dUTP ^* represents labeled dUTP and ddATP represents dideoxyadenosine triphosphate. Preferably, dUTP is
Each of the four amplicons is labeled with a separate spectrally resolvable fluorescent dye. The free tag (SEQ ID NO: 15) for each amplicon is mixed and applied to a spatially controllable array for hybridization to its complement and detection.

Example 4 Sequencing of a Target Polynucleotide with Conversion to RNA In this example, a dsDNA template is sequenced using rolling primers, according to an embodiment using cyclic conversion of the template to RNA. The following vector (SEQ ID NO: 16) was prepared from a standard cloning vector (eg, pUC19) using a T7 promoter element (double underlined) and a rolling primer binding site (single underlined).
By inserting at the indicated restriction site of the polylinker region:

[0102]

[Table 17] After insertion of the target polynucleotide into the BamHI site, the starting template (200) of FIG. 2a is obtained. After linearization by cutting the vector with HinD III, RNA transcription occurs.
Figures 2a and 2b show the changes that occur in the sequence of the template and rolling primer binding sites when the process is run for 8 cycles. In each cycle, one nucleotide in the template is identified. Arrow (21
0) indicates the nucleotide position at which the mutation occurs, and the double underlined nucleotide of "converted template" indicates the change that occurs. In this example, the p1 and p2 primers described above, with the indicated extension regions, are used with reverse transcriptase (Promega, Madison, Wis.). The lower case "a" in the extension region indicates oxo-A and the lower case "g" in the extension region indicates oxo-G. The amplification step is performed using PCR using a forward primer (shown below) containing a T7 promoter element (underlined) and the following p1 and p2 reverse primers:

[0103]

[Table 18] RNA is generated from dsDNA templates using the RiboMax RNA production system (Promega, Madison, WI) using the manufacturer's protocol. Briefly, 50 μl
In a reaction volume, 0.1 pmol of dsDNA template was added to 30 U /
μl T7 RNA polymerase, 1.5 U / μl human placental ribonuclease inhibitor, and 19 U / μl inorganic pyrophosphatase, and mix at 37 ° C. for 2-4 hours,
Transfer buffer (80 mM HEPES-KOH (pH 7.5), 2.4 mM MgC
l _2, 2 mM spermidine -HCl, 40 mM DTT, and each 7.5
(4 mM ribonucleoside triphosphate). After adding 47 μl H ₂ O and 1 μl of 100 mM MnCl ₂ and heating at 65 ° C. for 5 minutes, 2 μl (4.2 U) of DNas
Add eI (US Biochemical) and bring the mixture to 37 ° C
And incubate for 30 minutes. The RNA is then purified from the reaction mixture using the QIAGEN (Santa Clarita, CA) RNA purification system (30 μl elution volume).

[0104] Four separate reverse transcription reaction mixtures are formed. Each is a 1-10 pmol aliquot of the transcribed RNA and 5 pmol of fluorescein (eg, FAM (Perkin-El
mer Corp. Applied Biosystems Division, Foster City,
It contains the appropriate rolling primer labeled with a) available from CA). After heating to 65 ° C. for 5 minutes to denature the RNA, the reaction mixture was cooled on ice and reverse transcriptase (0.1 U / μl) and RNase inhibitor (0.
85 U / μl) with 50 mM Tris-HCl (pH 8.1), 8 mM MgCl ₂ , 5
Add to a buffer consisting of 0 mM NaCl, 10 mM DTT, and 4 deoxynucleoside triphosphates of 25-50 μM each, resulting in a reaction volume of 10 μl. Add 50 reaction mixture
Incubate at ~ 55 ° C for 5 minutes, then
Incubate at 5 ° C for 5 minutes, thereby effectively destroying any remaining RNA. The four reaction mixtures are
It corresponds to a rolling primer with 3 'ends A, C, G, and T, respectively. Thus, in each cycle, only one of the four reactions results in the synthesis of an ssDNA extension product. The products are identified by separating the reaction components by gel electrophoresis, after which the band containing the extension product is excised and the ssDNA is recovered.

Conventional methods using the primers listed above
By amplifying the ssDNA extension product in PCR, dsDN
A Re-form the template.

Example 5 dNTP Concentration for Primer Selection on RNA Template
In this example, the effect of different deoxynucleoside triphosphate concentrations on primer selection was determined in three cycles.
Investigated via RNA synthesis, primer selection, and amplification. The steps of each cycle were performed as described in Example 4, except that a mixture of each of the four primers was used in the extension reaction. As a result, primers having 3 'ends A, C, G, and T competed with each other for extension by reverse transcriptase at the indicated concentrations of dNTPs. Figures 5a-5c
Show that concentrations of dNTPs of about 50 μM or less lead to maximum selectivity in primer extension by reverse transcriptase. For FIG. 5a, the exact primers were as follows:

[0107]

[Table 19] For FIG. 5b, the exact primers were as follows:

[0108]

[Table 20] For FIG. 5c, the exact primers were as follows:

[0109]

[Table 21]

[0110]

The present invention provides a novel method and approach for determining the sequence of a polynucleotide.

[0111]

[Sequence list]

SEQUENCE LISTING <110> Lynx Therapeutics, Inc. <120> DNA Extension and Analysis with Rolling Prim
ers <130> F198566721 <150> US 08 / 916,120 <151> 1997-08-22 <160> 19 <170> PatentIn version 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 1 GGAAGAGGAA GAGGAAGANN NN 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 2 GAAGAGGAAG AGGAAGAGNN NN 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 3 AAGAGGAAGA GGAAGAGGNN NN 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 4 AGAGGAAGAG GAAGAGGANN NN 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 5 GAGGAAGAGG AAGAGGAANN NN 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 19..22 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 6 AGGAAGAGGA AGAGGAAGNN NN 22 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 22..26 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<220><221> modified base <222> 6 <223> / note =" N is a deoxyinosine. "<400> 7 GGGGGNGTGT GTTTTTGGGG GNNNNN 26 <210> 8 <211> 26 <212 > DNA <213> Artificial Sequence <220><221> modified base <222> 22..26 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<220><221> modified base <222> 6..12 <223> / note =" N is a deoxyinosine. "<400> 8 GGGGGNTGTG TNTTTTGGGG GNNNNN 26 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 22..25 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 9 CCTTCTCCTT CTCCTTCTTC CNNNN 25 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220><223> primer binding region <400> 10 AGTGGCTGGG CATCGGACCG 20 <210 > 11 <211> 20 <212> DNA <213> Artificial Sequence <220><223> primer binding region <400> 11 GGGGCCCAGT CAGCGTCGAT 20 <210> 12 <211> 62 <212> DNA <213> Artificial Sequence <220 ><223> vector <400> 12 AAAAGGAGGA GGCCTTGATA GAGAGGACCT GTTTAAACGG ATCCGCTGCT TTTTCCTCCT 60 CC 62 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 1..8 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 13 NNNNNNNNAA AAGGAGGAGG CCTTGA 26 <210> 14 <211> 43 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 1..8 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reduc
innucleotide, such as deoxyinosine. "<400> 14 NNNNNNNNAG AGAGAGAGAG GAGAGAGAGA GTAGAGAGGA CCG 43 <210> 15 <211> 12 <212> RNA <213> Artificial Sequence <220><223> released tag <400> 15 AUCUCUCCUG GC 12 <210> 16 <211> 104 <212> DNA <213> Artificial Sequence <220><223> vector <400> 16 AATTTAATAC GACTCACTAT AGGGAGAATT CGAGCTCGGT ACCCGGGGAT CCCCCCCAAA 60 AACACACACC CCCGTCGACC TGCAGGCATG CAAGCTTGGC GTAA 104 <210> 17 <211> 47 212> DNA <213> Artificial Sequence <220><223> primer <400> 17 AATTTAATAC GACTCACTAT AGGGAGAATT CGAGCTCGGT ACCCGGG 47 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 1..19 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 18 NGNGGNGTGT NTTTTTNGNG G 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220><221> modified base <222> 1..19 <223> / note = "N is a terminal nucleotide of either
A, C, G, or T, or a complexity-reducing nucleotid
e, such as deoxyinosine. "<400> 19 NGNGGNTGTG TNTTTTNGNG G 21

[Brief description of the drawings]

FIG. 1 is a schematic diagram illustrating the steps of a preferred embodiment using RNA template selection.

FIG. 2a is a schematic diagram illustrating the steps of a preferred method of the invention using simultaneous analysis of multiple tagged polynucleotides.

FIG. 2b is a schematic diagram illustrating the extended region of the rolling primer for a subsequent step selected based on the identification of the rolling primer extended region of the current step.

FIG. 3 is a schematic diagram illustrating an apparatus for detecting labeled tags on a spatially controllable array of tag complements.

FIG. 4a is a schematic diagram illustrating how the sequencing template changes in successive steps of a preferred embodiment of the present method.

FIG. 4b is a schematic diagram illustrating how the sequencing template changes in successive steps of a preferred embodiment of the method.

FIG. 5a shows dNTPs in the selectivity of rolling primer extension on RNA templates by reverse transcriptase.
It is a schematic diagram explaining the influence of density.

FIG. 5b shows dNTPs in the selectivity of rolling primer extension on RNA templates by reverse transcriptase.
It is a schematic diagram explaining the influence of density.

FIG. 5c shows dNTPs in the selectivity of rolling primer extension on RNA templates by reverse transcriptase.
It is a schematic diagram explaining the influence of density.

[Explanation of symbols]

 Reference Signs List 10 double-stranded DNA 12 primer binding site 14 cleavage site 16 tag 18 cleavage site 20 target polynucleotide 22 rolling primer binding site 24 extended region 28 container 30 container 32 container 34 container 38 amplicon A 40 amplicon C 42 amplicon G 44 Amplicon T 48 solid support 210 nucleotide position at which mutation occurs 500 flow chamber 502 inlet 504 outlet 506 glass plate 510 cavity 512 glass coverslip 514 syringe pump 522 valve block 524 irradiation light 526 light source 528 filter 530 fluorescence 532 confocal microscope 534 Filter 536 CCD camera 538 Workstation

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] December 4, 1998

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2a

FIG. 2b

FIG. 3

FIG. 4a

FIG. 5a

FIG. 5b

FIG. 4b

FIG. 5c

 ──────────────────────────────────────────────────続 き Continuation of the front page (71) Applicant 598114930 3832 Bay Center Place, Hayward, California 94545 U.S.A. S. A.

Claims (12)

[Claims] 1. A method for determining the nucleotide sequence of a polynucleotide, comprising: (a) providing a set of primers, wherein each primer of the set comprises a terminal nucleotide, a template arrangement. (B) forming a primer-binding site and a template containing the polynucleotide, wherein the primer-binding site comprises a set and an extended region containing one or more complexity-reducing nucleotides; Is complementary to at least one primer of
Amplifying the double-stranded DNA selectively formed by extending the primers from the set, wherein the extension region forms a duplex perfectly matched with the primer binding site of the template; (D) identifying the terminal nucleotide of an extension region of the primer by identifying the amplicon; (e) mutating the primer binding site of the template to Shifting the binding site by one nucleotide in the direction of extension; and (f) repeating steps (c)-(e) until the nucleotide sequence of the polynucleotide is determined. 2. The method of claim 1, wherein said amplicon is formed by amplifying said double-stranded DNA by polymerase chain reaction. 3. The method according to claim 2, wherein the reduced complexity nucleotide is deoxyinosine and the polymerase chain reaction and the elongation to form the double-stranded DNA are performed with deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyinosine triphosphate. 3. The method of claim 2, wherein the method is performed in the presence of, and thymidine triphosphate. 4. The step of mutating the primer binding site of the template includes the step of using the double-stranded DNA as the primer, wherein the template-located segment is adjacent to the primer binding site of the double-stranded DNA. 4. The method according to claim 3, wherein the step is performed by extending and amplifying with a primer containing the nucleotide mismatched with the nucleotide so that the identity of the adjacent nucleotide is changed by oligonucleotide-directed mutagenesis using the primer. Method. 5. A method for determining the nucleotide sequence of a polynucleotide, comprising: (a) providing a first set of primers, wherein each first primer of each of the sets comprises 3 primers. Having an '-terminal nucleotide, a template-located segment, and an extension region containing one or more complexity-reducing nucleotides; (b) a first primer binding site, a promoter, a polynucleotide, and a second primer binding site. Providing a double-stranded DNA template comprising: (c) wherein said first primer binding site is capable of forming an extendable duplex with at least one first primer; Producing a population of RNA transcripts from the double-stranded DNA template using an RNA polymerase that recognizes a promoter; (d) with it Mutating a first primer binding site in the RNA transcript by extending a first primer that forms an extendable duplex, such that the first primer binding site 1 nucleotide in the direction of
A step in which a template is formed; (e) the single-stranded DNA
Forming an amplicon from the template;
(F) identifying the 3 ′ terminal nucleotide of the first primer extended to form the single-stranded DNA template by identifying the amplicon; (g) determining the nucleotide sequence of the polynucleotide Repeating steps (b)-(f) until the process is completed. 6. The method of producing a population of RNA transcripts further comprises removing DNA from the population.
The method of claim 5. 7. The method of claim 6, wherein said amplicon is formed by amplifying said double-stranded DNA by a polymerase chain reaction. 8. The method according to claim 1, wherein one or more of the reduced complexity nucleotides of the extension region of the first primer comprises 2′-deoxyinosine, 8-oxo-2′-deoxyadenosine, and 8-oxo-2′-deoxy. The method of claim 7, wherein the method is selected from the group consisting of guanosine. 9. The method of removing DNA from the population of RNA transcripts comprises treating the population with DNase.
The method according to claim 8. 10. The method of claim 9, wherein said RNA polymerase is T7 RNA polymerase. 11. A oligonucleotide primers defined by the _{formula: 5'-X 1 X 2 ...} X k IRZNN wherein: X ₁ X ₂ ... X _k, each Xi is i = 1,2, ... k, and Xi is an oligo such as selected from the group consisting of 2'-deoxyadenosine, 2'-deoxycytosine, 2'-deoxyguanosine, thymidine, and 2'-deoxyinosine. I is 2'-deoxyinosine; R is selected from the group consisting of diaminopurine, 2'-deoxyguanosine, and thymidine; Z is 8-oxo
N is selected from the group consisting of -2'-deoxyadenosine and 8-oxo-2'-deoxyguanosine; N is selected from the group consisting of 2'-deoxyadenosine, 2'-deoxycytosine, 2'-deoxyguanosine, and thymidine And k
Is an oligonucleotide primer in the range of 8-30. 12. The oligonucleotide primer according to claim 11, wherein X ₁ X ₂ ... X _k are defined by the following formula: 5 ′-(G) _i I (GT) _j (T) _r (G) _m or 5 ′-(G) _i I (TG) _j (T) _r (G) _m where: G is 2′-deoxyguanosine, T is thymidine, and I is 2′- Deoxyinosine, i is 3
J is an integer between 3 and 5; r is an integer between 3 and 6; and m is an integer between 3 and 8.
An oligonucleotide primer that is an integer between