Sequencing a polynucleotide on a generic chip

Details Sequencing a polynucleotide on a generic chip Sequencing a polynucleotide on a generic chip

2000-07-20

2004-02-17

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S173100, C435S320100, C436S501000, C436S508000, C436S538000, C436S815000, C702S019000, C702S020000

Reexamination Certificate

active

06692915

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a method of analyzing a polynucleotide sequence. This invention is also directed to diagnostic aids for analyzing the nucleic acid composition and content of biological samples, including samples derived from medical and agricultural sources.
BACKGROUND OF THE INVENTION
The genomics revolution has spurned a significant interest in the creation of personalized drugs based on the genetic constitution, or ‘genotype’ of the recipient and in related scientific research areas such as high throughput drug discovery, diagnostics in human subjects, animal health and cloning, and the genetic selection of plants with desired traits. Consequently, robust technology platforms have been created for the rapid identification of genomics sequence and the creation of sequence databases wherefrom making electronic comparisons can identify even small differences in sequences between individuals (such as, single nucleotide polymorphisms, or SNPs). It is estimated that the ~80,000 human genes contain about 200,000 cSNPs, (i.e., SNPs that lie in coding regions) whereas over several million randomly chosen SNPs are likely to be detected in the genome.
It has been suggested that testing a dense panel of SNPs arrayed across both coding and noncoding regions, termed ‘ancestral haplotypes’, in affected individuals and in controls may be an appropriate strategy to identify associations that narrowly locate the neighborhood of a susceptibility or resistance gene. A dense map of SNPs with at least 100,000 SNPs is estimated to satisfy the resolution required.
Recent novel approaches to assessing DNA sequence differences between individuals, such as, minisequencing, multiplex reverse dot blots, DNA microarrays, and the TaqMan approach have lent a significant impetus to the speed, cost efficiency and accuracy of association studies.
Other strategies described in the art for identification of polymorphisms have included a genome simplification or complexity reduction step, including construction of shotgun libraries in BACs, PACs, or cosmids using DNA samples from a limited number of subjects. Clones from the library are sequenced. Alternatively, hybridization or enzymatic approaches (such as RDA or mismatch scanning using bacterial proteins or enzymes involved in homologous recombination) have been used. These latter approaches are rapid and cost effective and eliminate the need for sequencing extensive regions of genomic DNA for identifying polymorphisms.
Yet, the above methods suffer from 2 major disadvantages, a) lack of throughput, and b) inadequate representation of the genome per iteration. As such, there is a need for further improvements in the technology for high throughput genomics analytical methods, in particular, detection of SNPs.
SUMMARY OF THE INVENTION
This invention features methods for sensitively detecting the presence of a nucleic acid in a sample. Described herein are methods for synergistic multiplexed amplifications of nucleic acids. Multiple individual chemical and biochemical reactions for target identification, amplification and partitioning and detection of each signal independently of other similar signals in the multiplexed reaction can be caused to occur simultaneously.
Consequently, it is an object of this invention to provide a method for identifying nucleic acids in a sample.
It is an object of this invention to quantify nucleic acids from a sample.
It is an object of this invention to rapidly and cost-effectively determine the quantitative presence of nucleic acids in a sample.
It is an object of this invention to provide methods for the multiplexed analysis of nucleic acids from a sample.
It is an object of this invention to identify qualitative differences between nucleic acids of two or more organisms of the same species.
It is an object of this invention to provide a method for comparing the amount of nucleic acids present in two or more samples.
It is an object of this invention to identify an organism by identifying presence of the genomic nucleic acids of the organism in a sample.
It is an object of this invention to quantify the presence of nucleic acids of an organism relative to the amount of nucleic acids of another organism present in the same sample.
It is an object of this invention to identify the physiological state of an organism by quantifying gene expression in the sample.
In one aspect, the invention features a method of analyzing a polynucleotide, e.g., detecting a genetic event, e.g., a single nucleotide polymorphism, in a sample.
The method includes:
1. providing a sample which includes a sample polynucleotide sequence to be analyzed, said sample polynucleotide being at least partially single stranded and containing a region of recognizable sequence adjacent to the single stranded terminus;
2. (a) annealing an effective amount of sample sequence to a hairpin primer, wherein the hairpin primer comprises at least one copy of a nucleotide sequence complementary to the sequence of the sample polynucleotide sequence and optionally,
(b) extending the hairpin primer with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme to yield a product, e.g., a single stranded polymerase extended product complementary to a substantial portion of the single stranded region of the sample polynucleotide; and
(c) optionally, amplifying the polymerase extended product to produce amplified products, herein termed amplified products;
3. analyzing said product from 2 (a, b or c) by providing an array of a plurality of capture probes, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and wherein each positionally distinguishable capture probe includes at least one unique (i.e., not repeated in another capture probe) region, optionally, adjacent to a common (repeated in all the capture probes) region;
hybridizing the amplified products with the array of capture probes, thereby analyzing the sample sequence.
In preferred embodiments, analyzing a sample polynucleotide includes, e.g., sequencing the polynucleotide sequence, e.g., by sequencing by hybridization or positional sequencing by hybridization, detecting the presence of, or identifying a genetic event, e.g., a SNP, in a target nucleic acid, e.g., a DNA.
In preferred embodiments, the genetic event is within 12 bases, more preferably within 1, 2, 3, 4, 5, or 6 bases in the single stranded portion of sample polynucleotide that is adjacent to and abuts the double stranded region; more specifically, in the region of the single stranded portion of the sample polynucleotide farthest from the single stranded terminus, or is sufficiently close to the double strand portion of the sample polynucleotide that a mismatch would inhibit DNA polymerase-based extension from the sample polynucleotide/hairpin primer complex.
In preferred embodiments, the genetic event is located anywhere within the single stranded portion of the sample polynucleotide.
In preferred embodiments, the target polynucleotide in the sample is amplified, e.g., by PCR, prior to contact with a hairpin primer.
In preferred embodiments, the hairpin primer includes a site for a type 2s restriction enzyme and the site is positioned, e.g., such that a type 2s restriction enzyme binding at the site cleaves adjacent the region which binds the sample sequence or cleaves in the region which binds the sample sequence.
In a preferred embodiment, a region of the hairpin primer is complementary to a genetic event, e.g., a mutation or SNP, and hybridizes effectually to sample nucleic acid having the event and sample nucleic acid not having the event.
In preferred embodiments the polynucleotide sequence is: a DNA molecule: all or part of a known gene; wild type DNA; mutant DNA; a genomic fragment, particularly a human genomic fragment; a cDNA, particularly a human cDNA.
In preferred embodiments the polynucleotide sequence is: an RNA molecule: nucleic acids derived from RNA transcripts; wild type RNA; mu

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