Nucleic acid sequencing by raman monitoring of molecular deconstruction

Description Submit all comments and votes

FIELD OF THE INVENTION

The present methods, compositions and apparatus relate to the fields of molecular biology and genomics. More particularly, the methods, compositions and apparatus concern nucleic acid sequencing.

BACKGROUND

The advent of the human genome project required that improved methods for sequencing nucleic acids, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), be developed. Genetic information is stored in the form of very long molecules of DNA organized into chromosomes. The twenty-three pairs of chromosomes in the human genome contain approximately three billion bases of DNA sequence. This DNA sequence information determines multiple characteristics of each individual, such as height, eye color and ethnicity. Many common diseases, such as cancer, cystic fibrosis, sickle cell anemia and muscular dystrophy are based at least in part on variations in DNA sequence.

Determination of the entire sequence of the human genome has provided a foundation for identifying the genetic basis of such diseases. However, a great deal of work remains to be done to identify the genetic variations associated with each disease. That would require DNA sequencing of portions of chromosomes in individuals or families exhibiting each such disease, in order to identify specific changes in DNA sequence that promote the disease. RNA, an intermediary molecule required for processing of genetic information, can also be sequenced in some cases to identify the genetic bases of various diseases.

Existing methods for nucleic acid sequencing, based on detection of fluorescently labeled nucleic acids that have been separated by size, are limited by the length of the nucleic acid that can be sequenced. Typically, only 500 to 1,000 bases of nucleic acid sequence can be determined at one time. This is much shorter than the length of the functional unit of DNA, referred to as a gene, which can be tens or even hundreds of thousands of bases in length. Using current methods, determination of a complete gene sequence requires that many copies of the gene be produced, cut into overlapping fragments and sequenced, after which the overlapping DNA sequences may be assembled into the complete gene. This process is laborious, expensive, inefficient and time-consuming.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosed embodiments. The embodiments may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 illustrates an exemplary apparatus (not to scale) and method for DNA sequencing in which the released nucleotides are spatially separated from the nucleic acid molecule to be sequenced.

FIG. 2 illustrates an exemplary apparatus (not to scale) and method for DNA sequencing in which the released nucleotides are not spatially separated from the nucleic acid molecule. The detector quantifies the nucleotides present in solution.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods, compositions and apparatus are of use for the rapid, automated sequencing of nucleic acids. In particular embodiments, the methods, compositions and apparatus are suitable for obtaining the sequences of very long nucleic acid molecules of greater than 1,000, greater than 2,000, greater than 5,000, greater than 10,000 greater than 20,000, greater than 50,000, greater than 100,000 or even more bases in length. In various embodiments, such sequence information may be obtained during the course of a single sequencing run, using one molecule of nucleic acid 13, 102. In other embodiments, multiple copies of the nucleic acid molecule 13, 102 may be sequenced in parallel or sequentially to confirm the nucleic acid sequence or to obtain complete sequence data. In alternative embodiments, both the nucleic acid molecule 13, 102 and its complementary strand may be sequenced to confirm the accuracy of the sequence information. Advantages over prior methods of nucleic acid sequencing include the ability to read long nucleic acid sequences in a single sequencing run, greater speed of obtaining sequence data, decreased cost of sequencing and greater efficiency in terms of the amount of operator time required per unit of sequence data generated.

In certain embodiments, the nucleic acid 13, 102 to be sequenced is DNA, although it is contemplated that other nucleic acids 13, 102 comprising RNA or synthetic nucleotide analogs could be sequenced as well. The following detailed description contains numerous specific details in order to provide a more thorough understanding of the disclosed embodiments. However, it will be apparent to those skilled in the art that the embodiments may be practiced without these specific details. In other instances, devices, methods, procedures, and individual components that are well known in the art have not been described in detail herein.

In some embodiments, disclosed in FIG. 1 and FIG. 2, the methods involve sequencing of individual single-stranded nucleic acid molecules 13, 102 that are attached to an immobilization surface 14, 103 in a reaction chamber 11, 101 and disassembled in a deconstruction reaction. In such embodiments, the reaction chamber 11, 101 contains one or more deconstruction reagents 15, 106 that sequentially remove one nucleotide 16, 104 at a time from the unattached end 17, 105 of the nucleic acid molecule 13, 102. Non-limiting examples of such deconstruction reagents 15, 106 include any exonuclease known in the art. In some embodiments, the nucleotides 16, 104 are identified by Raman spectroscopy as they are released into solution.

Certain embodiments are illustrated in FIG. 1. FIG. 1 shows an apparatus 10 for nucleic acid sequencing comprising a reaction chamber 11 attached to a flow path 12. The reaction chamber 11 contains a nucleic acid molecule 13 attached to an immobilization surface 14 along with a deconstruction reagent 15, such as an exonuclease. The exonuclease 15 catalyzes the sequential release of individual nucleotides 16 from the free end 17 of the nucleic acid molecule 13. As the individual nucleotides 16 are released by the deconstruction reaction and enter solution, they move down the flow path 12 past a detection unit 18. The detection unit 18 comprises an excitation source 19, such as a laser, that emits an excitatory beam 20. The excitatory beam 20 interacts with the released nucleotides 16 so that electrons are excited to a higher energy state. The Raman emission spectrum that results from the return of the electrons to a lower energy state is detected by a Raman spectroscopic detector 21, such as a spectrometer or a monochromator.

In embodiments illustrated in FIG. 1, the released nucleotides 16 are spatially separated from the nucleic acid molecule 13 before detection by the detection unit 18. Spatial separation acts to increase the signal-to-noise ratio of the Raman detector 21 by isolating the individual nucleotides 16.

FIG. 1 illustrates embodiments in which a single nucleic acid molecule 13 is contained in a single reaction chamber 11. In alternative embodiments, multiple nucleic acid molecules 13, each in a separate reaction chamber 11, may be sequenced simultaneously. In such cases, the nucleic acid molecule 13 in each reaction chamber 11 may be identical or may be different. In other alternative embodiments, two or more nucleic acid molecules 13 may be present in a single reaction chamber 11. In such embodiments, the nucleic acid molecules 13 will be identical in sequence. Where more than one nucleic acid molecule 13 is present in the reaction chamber 11, the Raman emission signals will represent an average of the nucleotides 16 released simultaneously from all nucleic acid molecules 13 in the reaction chamber 11. The skilled artisan will be able to correct the signal obtained at any given time for deconstruction reactions that either lag behind or precede the majority of reactions occurring in the reaction chamber 11, using known data analysis techniques. In certain embodiments, the skilled artisan may use procedures to synchronize the deconstruction of multiple nucleic acid molecules 13 present in a single reaction chamber 11, as by adding a bolus of deconstruction reagents 15 with rapid mixing.

In certain alternative embodiments, a tag molecule may be added to the reaction chamber 11 or to the flow path 12 upstream of the detection unit 18. The tag molecule binds to and tags free nucleotides 16 as they are released from the nucleic acid molecule 13. This post-release tagging avoids problems that are encountered when the nucleotides 16 of the nucleic acid molecule 13 are tagged before their release into solution. For example, the use of bulky fluorescent probe molecules may provide considerable steric hindrance when each nucleotide 16 incorporated into a nucleic acid molecule 13 is labeled before deconstruction, reducing the efficiency and increasing the time required for the sequencing reaction.

In embodiments involving post-release tagging of nucleotides 16, it is contemplated that alternative methods of detection may be used, for example fluorescence spectroscopy or luminescence spectroscopy. Many alternative methods of detection of free nucleotides 16 in solution are known and may be used. For such methods, the Raman spectroscopic detection unit 18 may be replaced with a detection unit 18 designed to detect fluorescence, luminescence or other types of signals.

The tag molecules have unique and highly visible optical signatures that can be distinguished for each of the common nucleotides 16. In certain embodiments, the tag may serve to increase the strength of the Raman emission signal or to otherwise enhance the sensitivity or specificity of the Raman detector 21 for nucleotides 16. Non-limiting examples of tag molecules that could be used for embodiments involving Raman spectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine and aminoacridine. Other tag moieties that may be of use for particular embodiments include cyanide, thiol, chlorine, bromine, methyl, phosphorus and sulfur. In certain embodiments, carbon nanotubes may be of use as Raman tags. The use of tags in Raman spectroscopy is known in the art (e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677). The skilled artisan will realize that Raman tags should generate distinguishable Raman spectra when bound to different nucleotides 16, or different labels should be designed to bind only one type of nucleotide 16.

In certain embodiments, the nucleic acid molecule 13 is fixed in place, as by attachment to an immobilization surface 14, and immersed in a microfluidic flow down a flow path 12 that transports the released nucleotides 16 away from the nucleic acid molecule 13 and past a detection unit 18. In non-limiting examples, the microfluidic flow may result from a bulk flow of solvent past the nucleic acid molecule 13 and down a flow path 12, for example, a microcapillary tube or an etched channel in a silicon, glass or other chip. In alternative embodiments, the bulk medium moves only slowly or not at all, but charged species within the solution (such as negatively charged nucleotides 16) move down a flow path 12 comprising a channel or tube in response to an externally applied electrical field.

In other alternative embodiments, the n