The invention relates generally to novel methods and materials for nucleic acid sequence analysis by hybridization, in which the hybridization reaction occurs in a solution environment.
The rate of determining the sequence of the four nucleotides in nucleic acid samples is a major technical obstacle for further advancement of molecular biology, medicine, and biotechnology. Nucleic acid sequencing methods which involve separation of nucleic acid molecules in a gel have been in use since 1978. The other proven method for sequencing nucleic acids is sequencing by hybridization (SBH).
The traditional method of determining a sequence of nucleotides (i.e., the order of the A, G, C and T nucleotides in a sample) is performed by preparing a mixture of randomly terminated, differentially labeled nucleic acid fragments by degradation at specific nucleotides, or by dideoxy chain termination of replicating strands. Resulting nucleic acid fragments in the range of 1 to 500 bp are then separated on a gel to produce a ladder of bands wherein the adjacent samples differ in length by one nucleotide.
SBH does not require single base resolution in separation, degradation, synthesis or imaging of a nucleic acid molecule. Using mismatch discriminative hybridization of short oligonucleotides K bases in length, lists of constituent K-mer oligonucleotides may be determined for target nucleic acid. Sequence for the target nucleic acid may be assembled by uniquely overlapping scored oligonuclcotides.
There are several approaches available to achieve sequencing by hybridization. In a process called SBH Format 1, nucleic acid samples are arrayed, and labeled probes are hybridized with the samples. Replica membranes with the same sets of sample nucleic acids may be used for parallel scoring of several probes and/or probes may be multiplexed. Nucleic acid samples may,be arrayed and hybridized on nylon membranes or other suitable supports. Each membrane array may be reused many times. Format 1 is especially efficient for batch processing large numbers of samples.
In SBH Format 2, probes are arrayed at locations on a substrate which correspond to their respective sequences, and a labeled nucleic acid sample fragment is hybridized to the arrayed probes. In this case, sequence information about a fragment may be determined in a simultaneous hybridization reaction with all of the arrayed probes. For sequencing other nucleic acid fragments, the same oligonucleotide array may be reused. The arrays may be produced by spotting or by in situ synthesis of probes.
In Format 3 SBH, two sets of probes are used. In one embodiment, a set may be in the form of arrays of probes with known positions, and another, labeled set may be stored in multiwell plates. In this case, target nucleic acid need not be labeled. Target nucleic acid and one or more labeled probes are added to the arrayed sets of probes. If one attached probe and one labeled probe both hybridize contiguously on the target nucleic acid, they are covalently ligated, producing a detected sequence equal to the sum of the length of the ligated probes. The process allows for sequencing long nucleic acid fragments, e.g. a complete bacterial genome, without nucleic acid subcloning in smaller pieces.
However, to sequence long nucleic acids unambiguously, SBH involves the use of long probes. As the length of the probes increases, so does the number of probes required to generate sequence information. Each 2-fold increase in length of the target requires a one-base increase in the length of the probe, resulting in a four-fold increase in the number of probes required (the complete set of all possible sequences of probes of length k contains 4k probes). For example, sequencing 100 bases of DNA requires 16,384 7-mers; sequencing 200 bases requires 65,536 8-mers; 400 bases, 262,144 9-mers; 800 bases, 1,048,576 10-mers; 1600 bases, 4,194,304 11-mers; 3200 bases, 16,777,216 12-mers; 6400 bases, 67,108,864 13-mers; and 12,800 bases requires 268,435,456 14-mers.
Because a limited number of probes can be scored in each array-based hybridization reaction, use of an extremely large number of probes requires carrying out multiple hybridization reactions.
An improvement in SBH that increases efficiency and reduces the number of hybridization reactions would greatly enhance the practical ability to sequence long pieces of polynucleotides de novo. Such an improvement would, of course, also enhance resequencing and other applications of SBH. Thus, there remains a need for additional and improved methods and materials for performing sequence analysis by hybridization.
The present invention provides novel methods and materials, including apparatus and kits, for performing sequence analysis by hybridization (referred to herein as xe2x80x9cSBHxe2x80x9d). According to the present invention, the efficiency, sensitivity and accuracy of these methods is improved by performing the entire hybridization step in solution, preferably coupled with single probe molecule detection. The methods and materials of the present invention advantageously allow for easier preparation of probes without attaching them to a fixed support, allow the use of larger numbers and different types of probes, improve hybridization and enzymatic kinetics relative to solid-phase hybridization (when either target or probe(s) are bound to a solid support), and allow for use of a different range of detection devices.
In one aspect, the invention provides methods of detecting a sequence of a target nucleic acid, comprising: (a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe:target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support; (b) detecting probe molecules that hybridize with the target nucleic acid, using a reader capable of detecting an individual probe molecule; and (c) detecting a sequence of the target nucleic acid by overlapping sequences of the information regions of at least two of the probe molecules contacted with the target in step (a). Methods of the invention are carried out wherein at least two mixtures are contacted simultaneously, or alternatively wherein at least two mixtures are contacted sequentially. Methods of the invention include those wherein at least about 10 probe molecules distinct in their information regions, at least about 100 probe molecules distinct in their information regions, at least about 1,000 probe molecules distinct in their information regions, or at least about 10,000 probe molecules distinct in their information regions. In one aspect, methods of the invention include probe molecules that comprise modified bases.
Multiple probe molecules of the invention may also be associated with identification tags, and in one aspect, multiple probe molecules each have two identification tags. In one aspect, methods may include multiple probe molecules having the same information region which are each associated with the same identification tag. In another aspect, at least two probe molecules having different information regions are associated with different identification tags.
Methods of the invention include those wherein the probe molecules are divided into pools, wherein each pool comprises at least two probe molecules having different information regions, and all probe molecules within each pool are associated with the same identification tag which is unique to the pool. In one aspect, at least one identification tag is a bar code. Methods are provided wherein the bar code is based on a property selected from the group consisting of size, shape, electrical properties, magnetic properties, optical properties, and chemical properties. Alternatively, the identification tag is a DNA bar code comprising modified bases, a molecular bar code, or a nanoparticle bar code. In one aspect, the bar code comprises elements of varying length, each element comprising a preset number of unit tags. The preset number may vary, e.g., may be 1, 2, 3, 4, 5 or more depending on the desired number of combinations and the type of unit tags.
In another aspect, methods of the invention include a target nucleic acid which is associated with a separator tag. Alternatively, methods are provided wherein the probe molecules are associated with separator tags.
The invention further provides methods wherein before the detection step (b) described above, probe molecules that hybridize to the target nucleic acid are separated from probe molecules that do not hybridize to the target nucleic acid. In one aspect, probe molecules that do not hybridize to the target nucleic acid are eliminated by enzymatic digestion.
The invention also provides methods wherein step (b) further comprises counting the number of times probe molecules having the same information region are detected. In one aspect, the methods of the invention include a reader comprising a nanopore channel which is used to detect probe molecules in step (b). Alternatively, methods include sensing of electrical responses within or around the nanopore channel is used to detect probe molecules in step (b). In one aspect, the reader detects molecular bar codes in step (b).
The invention further provides methods wherein the probe molecules are associated with one or more tags that allow identification of 5xe2x80x2/3xe2x80x2 orientation of probe molecules during detection step (b). In another embodiment, methods of the invention, the sequence of the probe molecule(s) is detected in step (b). In one aspect, methods are provided wherein at least two probe molecules are associated with identification tags and the identification tags are also detected in step (b).
The invention further provides methods of sequencing a target nucleic acid, comprising: (a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe:target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support; (b) covalently joining probe molecules that form contiguous probe:target hybrids that are perfectly complementary to the target in the information region of the probe molecules; and (c) detecting covalently joined probe molecules, using a reader capable of detecting an individual probe molecule. In another aspect, methods of the invention further comprise the step of: (d) detecting a sequence of the target nucleic acid by overlapping at least two sequences generated by combining sequences of the information region of two probe molecules contacted with target nucleic acid in step (a). As used herein, xe2x80x9ccombining sequences of the information region of two probe moleculesxe2x80x9d means contiguously combining sequences in proper 5xe2x80x2-3xe2x80x2 orientation. In another embodiment, methods are provided wherein before detection step (c), covalently joined probe molecules are separated from probe molecules that have not been covalently joined.
Also provided are methods of the invention wherein at least one nucleotide is added to the end of one or more probe molecules that hybridize to target nucleic acid using a polymerase or active fragment thereof. In one aspect, the probe molecules are contacted with a mixture of four different uniquely labeled nucleotides.
Methods are provided wherein target nucleic acids comprising an entire human genome are contacted with probe molecules. Alternatively, methods are provided wherein a single nucleotide polymorphism is detected.
The invention further provides kits comprising a mixture of probe molecules, wherein about 100 or more probe molecules each have distinct information regions, wherein two or more of the sequences of said distinct information regions within the mixture overlap. In one aspect, the kits include about 105 or less probe molecules each have the same information region. In another embodiment, the kits include about 104 or less probe molecules each have the same information region. Alternatively, kits of the invention include those wherein each information region is represented by 104 or more probe molecules having the same information region. Also provided are kits wherein at least two probe molecules having the same information region have the same identification tag.
In one aspect, kits are provided comprising a set of mixtures of probe molecules, wherein about 100 or more probe molecules each have distinct information regions, wherein two or more of the sequences of said distinct information regions within the set overlap. In one aspect, kits of the invention are provided wherein about 105 or less probe molecules each have the same information region. In another aspect, kits are provided wherein at least two probe molecules having different information regions are in the same pool and have the same identification tag. Kits are also provided wherein about 5000 or more probe molecules each have the same information region.
The invention further provides tags which are bar codes comprising an alternating arrangement of elements of varying detectable properties, wherein consecutive elements have a difference in at least one of the detectable properties. In one aspect, the elements in the tag comprise multiple unit tags of varying detectable properties and said elements vary in length.
The present invention provides methods for analyzing the sequence of a target nucleic acid, comprising the steps of (a) contacting a target nucleic acid with a mixture of a plurality of oligonucleotide probes (which may include a plurality of probe molecules) of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region, under conditions which discriminate between probe:target hybrids that are perfectly complementary in the information region of the probe and probe:target hybrids that are mismatched in the information region of the probe (i.e., under conditions which produce, on average, more probe:target hybridization with probes which are perfectly complementary to the target nucleic acid in the information region of the probes than with probes which are mismatched in the information regions), wherein the target nucleic acid is not attached to a fixed support, and wherein the probes are not attached to a fixed support; (b) detecting a subset of probes that hybridize with the target nucleic acid, preferably using a reader capable of detecting an individual probe molecule; and (c) determining the sequence of the target nucleic acid from two or more of the probes detected in step (b).
Depending on the conditions of hybridization and the use of pooling methods, described in further detail below, step (b) may include detection of more probes than the subset that hybridizes with the target nucleic acid. However, the SBH process and algorithms are very robust and can handle a large number of false positive probes, as discussed more fully in U.S. Provisional Application Ser. No. 60/115,284 filed Jan. 6, 1999, and related co-owned, co-pending U.S. application Ser. No. 09/479,608 filed Jan. 6, 2000, both of which are incorporated herein by reference.
Determining the sequence in step (c) can be done, for example, by actually overlapping the sequences of some (e.g., two or more, three or more, or four or more) or all of the detected probes for the target nucleic acid, or by comparing the detected set of probes for the target nucleic acid (which may be correlated with the identity of a nucleic acid sample to serve as a signature for identifying the nucleic acid sample) to the detected set for another target nucleic acid.
Optionally, between steps (a) and (b), a step of separating probe:target hybrids that are perfectly complementary in the information region of the probe from probe:target hybrids that are mismatched in the information region of the probe is carried out. Alternatively, between steps (a) and (b), a step of covalently joining probes that form contiguous probe:target hybrids that are perfectly complementary to the target in the information region of the probes is carried out, and in step (b) a subset of covalently joined probes is detected.
The probes may be associated with identification tags. Each of the probes may be associated with a unique identification tag; alternatively, the probes may be divided into informational pools, and all probes within each informational pool are associated with an identification tag unique to the informational pool. A preferred identification tag is a DNA bar code. The target nucleic acid or the probes may also be associated with one or more separator tags that aid separation of the probe:target hybrids from the unhybridized nucleic acids.
The number of probes used in the hybridization step may be at least about 10, at least about 100, at least about 1000, at least about 104, at least about 105, at least about 106, or at least about 107 different probes (meaning the number of probe sequences distinct in their information regions), and may potentially range up to about 1010 different probes or even more. According to this method, the mixture of probe molecules comprises at least two and preferably many more probe molecules each having different information regions.
In detection step (b), the positive probes may be detected using a reader comprising a nanopore channel. Detection may take place via, e.g., sensing of electrical responses within or around the nanopore channel as the probe molecule passes through or over the pore. In preferred embodiments of the method, a reader comprising a nanopore channel detects a DNA bar code associated with each probe, or detects the sequence of the probe itself. Alternatively, the positive probes may be detected using any suitable reader known in the art that is capable of detecting single probe molecules.
Another aspect of the invention provides an apparatus comprising means for carrying out the hybridization step and means for carrying out the detecting step, as described above. Such an apparatus preferably comprises a reader comprising a nanopore channel.
A further aspect of the invention provides sets of probes in the form of one or more kits, each set comprising a mixture of different probes. The set of probes may comprise at least about 10, 100, 1000, 104, 105, 106, 107, 108, 109, or 1010 different probes (meaning the number of probe sequences distinct in their information region). Preferably each information region is represented by about 104 or more probe molecules (which may include degenerate ends). Each probe in the set may be associated with one or more identification tags and optionally one or more separator tags. Each probe in the set need not be associated with a unique identification tag, particularly if the kit is intended for use with pooling methods in SBH.
The improved SBH efficiency provided by the present invention is particularly advantageous for sequencing and resequencing applications that require an extremely large number of probes. Examples of applications that require very large numbers of probes are: (1) sequencing or resequencing of the entire human genome and other complex genomes, (2) sequencing or resequencing of total mRNA or cDNA in a human or other complex cell, (3) genotyping thousands or millions of single nucleotide polymorphisms in individual human genomes, (4) de novo sequencing of thousands of bases. Potentially, all of the probes that would be needed to perform these types of sequence analysis could be used in a single solution-based hybridization reaction with the target polynucleotide sequence.
The methods and materials of the invention also may be useful for carrying out DNA computing, described, for example, in Ouyang et al., Science, 278:446-9 (1997), Guarnieri et al., Science, 273:220-3 (1996).
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.