Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-27
2003-03-25
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091520, C536S023100, C536S024320
Reexamination Certificate
active
06537755
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND
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 4
k
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.
SUMMARY OF THE INVENTION
The present invention provides novel methods and materials, including apparatus and kits, for performing sequence analysis by hybridization (referred to herein as “SBH”). 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
Chakrabarti Arun Kr.
Fredman Jeffrey
Marshall Gerstein & Borun
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