Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-06
2004-11-16
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100
Reexamination Certificate
active
06818395
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for high speed, high throughput analysis of polynucleotide sequences and apparatuses for carrying out such methods.
BACKGROUND OF THE INVENTION
Traditional DNA sequencing techniques share three essential steps in their approaches to sequence determination. First, a multiplicity of DNA fragments are generated from a DNA species which it is intended to sequence. These fragments are incomplete copies of the DNA species to be sequenced. The aim is to produce a ladder of DNA fragments, each a single base longer than the previous one. For example, with the Sanger method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463, 1977), the target DNA is used as a template for a DNA polymerase to produce a number of incomplete clones. These fragments, which differ in respective length by a single base, are then separated on an apparatus which is capable of resolving single-base differences in size. The third and final step is the determination of the nature of the base at the end of each fragment. When ordered by the size of the fragments which they terminate, these bases represent the sequence of the original DNA species.
Automated systems for DNA sequence analysis have been developed, such as discussed in Toneguzzo et al., 6 Biotechniques 460, 1988; Kanbara et al., 6 Biotechnology 816, 1988; and Smith et al., 13 Nuc. Acid. Res. 13: 2399, 1985; U.S. Pat. No. 4,707,237 (1987). However, all these methods still require separation of DNA products by a gel permeation procedure and then detection of their locations relative to one another along the axis of permeation or movement through the gel. These apparatuses used in these methods are not truly automatic sequencers. They are merely automatic gel readers, which require the standard sequencing reactions to be carried out before samples are loaded onto the gel.
The disadvantages of the above methods are numerous. The most serious problems are caused by the requirement for the DNA fragments to be size-separated on a polyacrylamide gel. This process is time-consuming, uses large quantities of expensive chemicals, and severely limits the number of bases which can be sequenced in any single experiment, due to the limited resolution of the gel. Sanger dideoxy sequencing has a read length of approximately 500 bp, a throughput that is limited by gel electrophoresis (appropriately 0.2%).
Other methods for analyzing polynucleotide sequences have been developed more recently. In some of these methods broadly termed sequencing by synthesis, template sequences are determined by priming the template followed by a series of single base primer extension reactions (e.g., as described in WO 93/21340, WO 96/27025, and WO 98/44152). While the basic scheme in these methods no longer require separation of polynucleotides on the gel, they encounter various other problems such as consumption of large amounts of expensive reagents, difficulty in removing reagents after each step, misincorporation due to long exchange times, the need to remove labels from the incorporated nucleotide, and difficulty to detect further incorporation if the label is not removed. Many of these difficulties stem directly from limitations of the macroscopic fluidics employed. However, small-volume fluidics have not been available. As a result, these methods have not replaced the traditional gel-based sequencing schemes in practice. The skilled artisans are to a large extent still relying on the gel-based sequencing methods.
Thus, there is a need in the art for methods and apparatuses for high speed and high throughput analysis of longer polynucleotide sequences which can be automated utilizing the available scanning and detection technology. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
In one aspect of the present invention, methods for analyzing the sequence of a target polynucleotide are provided. The methods include the steps of (a) providing a primed target polynucleotide linked to a microfabricated synthesis channel; (b) flowing a first nucleotide through the synthesis channel under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide; (c) determining presence or absence of a signal, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide; (d) removing or reducing the signal, if present; and (e) repeating steps (b)-(d) with a further nucleotide that is the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer.
In some methods, step (a) comprises providing a plurality of different primed target polynucleotides linked to different synthesis channels; step (b) comprises flowing the first nucleotide through each of the synthesis channels; and step (c) comprises determining presence or absence of a signal in each of the channels, the presence of a signal in a synthesis channel indicating the first nucleotide was incorporated into the primer in the synthesis channel, and hence the identity of the complementary base that served as a template in the target polynucleotide in the synthesis channel. In some methods, a plurality of different primed target polynucleotides are linked to each synthesis channels.
Some methods include the further steps of flushing the synthesis channel to remove unincorporated nucleotides. In some methods, steps (b)-(d) are performed at least four times with four different types of nucleotides. In some methods, steps (b)-(d) are performed until the identity of each base in the target polynucleotide has been identified.
In some methods, the nucleotides are labeled. The label can be a fluorescent dye, and the signal can be detected optically. The label can also be a radiolabel, and the signal can be detected with a radioactivity detector. In some methods, incorporation of nucleotides is detected by measuring pyrophosphate release.
In some methods, the synthesis channel is formed by bonding a microfluidic chip to a flat substrate. In some of these methods, the target polynucleotides are immobilized to the interior surface of the substrate in the synthesis channel. In some of these methods, the interior surface is coated with a polyelectrolyte multilayer (PEM). In some of these methods, the microfluidic chip is fabricated with an elastomeric material such as RTV silicone.
In another aspect of the present invention, methods for analyzing a target polynucleotide entails (a) pretreating the surface of a substrate to create surface chemistry that facilitates polynucleotide attachment and sequence analysis; (b) providing a primed target polynucleotide attached to the surface; (c) providing a labeled first nucleotides to the attached target polynucleotide under conditions whereby the labeled first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide; (d) determining presence or absence of a signal from the primer, the presence of a signal indicating that the labeled first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide; and (e) repeating steps (c)-(d) with a labeled further nucleotide that is the same or different from the first labeled nucleotide, whereby the labeled further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer.
In some of these methods, the substrate is glass and the surface is coated with a polyelectrolyte multilayer (PEM). In some methods, the PEM is terminated with a polyanion. In some methods, the polyanion is terminated with carboxylic acid groups. In some methods, the target polynucleotide is biotinylated, and the PEM-coated surface is further coated with biotin and then streptavidin.
In still another aspect of the presen
Quake Stephen
Unger Marc
California Institute of Technology
Chakrabarti Arun
Fredman Jeffrey
Proskauer Rose LLP
LandOfFree
Methods and apparatus for analyzing polynucleotide sequences does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and apparatus for analyzing polynucleotide sequences, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and apparatus for analyzing polynucleotide sequences will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3292979