Sequencing by incorporation

Details Sequencing by incorporation Sequencing by incorporation

2000-02-22

2003-09-02

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06613513

ABSTRACT:

BACKGROUND OF THE INVENTION
Most DNA sequencing today is carried out by chain termination methods of DNA sequencing. The most popular chain termination methods of DNA sequencing are variants of the dideoxynucleotide mediated chain termination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad. Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing, see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (e.g., Supplement 38, current through 1998) (Ausubel), Chapter 7. Thousands of laboratories employ dideoxynucleotide chain termination techniques. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. In addition to the Sanger methods of chain termination, new PCR exonuclease digestion methods have also been developed for DNA sequencing. Direct sequencing of PCR generated amplicons by selectively incorporating boronated nuclease resistant nucleotides into the amplicons during PCR and digestion of the amplicons with a nuclease to produce sized template fragments has been performed (Porteret al. (1997) Nucleic Acids Research 25(8):1611-1617). The above methods typically require that the terminated fragments be sequenced upon completion of the reaction. This is a time consuming step that limits the ability to sequence in a high throughput manner.
The development of microfluidic technologies by the inventors and their co-workers has provided a fundamental shift in how artificial biological and chemical processes are performed. In particular, the inventors and their co-workers have provided microfluidic systems that dramatically increase throughput for biological and chemical methods, as well as greatly reducing reagent costs for the methods. In these microfluidic systems, small volumes of fluid are moved through microchannels by electrokinetic or pressure-based mechanisms. Fluids can be mixed, and the results of the mixing experiments determined by monitoring a detectable signal from products of the mixing experiments.
Complete integrated systems with fluid handling, signal detection, sample storage and sample accessing are available. For example, Parce et al. “High Throughput Screening Assay Systems in Microscale Fluidic Devices” WO 98/00231 and Knapp et al. “Closed Loop Biochemical Analyzers” (WO 98/45481; PCT/US98/06723) provide pioneering technology for the integration of microfluidics and sample selection and manipulation. For example, in WO 98/45481, microfluidic apparatus, methods and integrated systems are provided for performing a large number of iterative, successive, or parallel fluid manipulations. For example, integrated sequencing systems, apparatus and methods are provided for sequencing nucleic acids. This ability to iteratively sequence a large nucleic acid (or a large number of nucleic acids) provides for increased rates of sequencing, as well as lower sequencing reagent costs. Applications to compound screening, enzyme kinetic determination, nucleic acid hybridization kinetics and many other processes are also described by Knapp et al.
New or improved methods of sequencing are accordingly desirable, particularly those that take advantage of high-throughput, low cost microfluidic systems. The present invention provides these and other features by providing new sequencing methods and high throughput microscale systems for providing sequencing reactions as well as many other features that will be apparent upon complete review of the following disclosure.
SUMMARY OF THE INVENTION
The present invention provides novel methods of sequencing by synthesis or incorporation. Nucleotides or nucleotide analogs are added to reaction mixtures comprising nucleic acid templates and primers, e.g., DNA or RNA. The nucleotides are incorporated into the primer, resulting in an extended primer. The sequence is determined as each additional complementary nucleotide is incorporated into the primer and the steps are repeated until the entire template sequence or a portion thereof is determined.
In one embodiment, the nucleotides or nucleotide analogs, or a fraction thereof, comprise a 3′-blocking group and a detectable label moiety, which typically comprises a phosphate or a carbamate group. The 3′-blocking groups provide reversible chain termination. When added to a growing nucleic acid chain, these nucleotide analogs result in a non-extendable primer. The 3′-blocking group is typically removed, e.g., by a reducing agent and/or a phosphatase, to produce an extendable primer to which further nucleotides are added, thereby allowing continued sequencing of the nucleic acid template. Removal of the 3′-blocking group is optionally performed before or after detection of the added nucleotide.
In another embodiment, the nucleotides or nucleotide analogs comprise a fluorescent label. Sequencing by synthesis using fluorescent nucleotides typically involves photobleaching the fluorescent label after detecting an added nucleotide. Photobleaching comprises applying a light pulse that destroys or reduces to an acceptable level, e.g., a background level or to a low enough level to prevent signal buildup over several sequencing cycles, the fluorescence of the nucleotides, e.g., a fluorescent nucleotide that has been added to the primer. The light pulse is typically applied for about 20 seconds or less, about 10 seconds or less, about 2 seconds or less, about 1 second or less, or about 0.1 second or less. The light pulse typically has a wavelength equal to the wavelength of light absorbed by the fluorescently labeled nucleotides. Detection of the added fluorescently labeled nucleotide occurs prior to or concurrent with photobleaching of the fluorescently labeled nucleotides and/or the extended primer. Nucleic acid templates comprising about 50 or more nucleotides, about 100 or more nucleotides, about 500 or more nucleotides, about 1000 or more nucleotides, about 2000 or more nucleotides, or about 10,000 or more nucleotides are optionally sequenced using these methods, e.g., sequenced with at least about 80%, at least about 90%, or at least about 95% accuracy.
In another embodiment, sequencing comprises sequencing by synthesis using detection of intercalating dyes (“sequencing by intercalation”). An intercalating dye is incubated or mixed with the template and primer as the sequencing reactions occur. When a nucleotide, e.g., a naturally occurring, non-labeled nucleotide, is incorporated into the primer, it forms an extended double-stranded region, into which intercalating dyes insert, e.g., between the stacked bases. The intercalating dye is detected, thus detecting the addition of a nucleotide to the growing chain and sequencing the template nucleic acid. The intercalating dye optionally comprises ethidium, ethidium bromide, an acridine dye, an intercalating nucleic acid stain, a cyanine dye, such as SYBR green, proflavin, propidium iodide, acriflavin, proflavin, actinomycin, anthracyclines, or nogalamycin. In some embodiments, photobleaching is performed after detecting the intercalating dye or approximately concurrent with detecting the intercalating dye.
The nucleotides or nucleotide analogs in the present invention typically comprise nucleoside 5′-triphosphates (dNTPs), e.g., deoxyadenosine 5′-triphosphate (dATP), deoxyguanosine 5′-triphosphate (dGTP), deoxycytidine 5′-triphosphate (dCTP), deoxythymidine 5′-triphosphate (dTTP), deoxyuridine 5′-triphosphate (dUTP), adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP), uridine 5′-triphosphate (UTP), or analogs thereof.
In some embodiments, the nucleotides or nucleotide analogs, or a fraction thereof, comprise a detectable label moiety, e.g., a fluorescent or chemiluminescent label moiety. Different nucleotides optionally comprise detectably different labels, e.g., ATP, GTP, CTP, TTP, and UTP each optional

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