High speed parallel molecular nucleic acid sequencing

Abstract

A method and device is disclosed for high speed, automated sequencing of nucleic acid molecules. A nucleic acid molecule to be sequenced is exposed to a polymerase in the presence of nucleotides which are to be incorporated into a complementary nucleic acid strand. The polymerase carries a donor fluorophore, and each type of nucleotide (e.g. A, T / U, C and G) carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal (but the laser wavelength does not stimulate the acceptor fluorophore). In particular embodiments, no laser is needed if the donor fluorophore is a luminescent molecule or is stimulated by one. The emission signal from the polymerase is capable of stimulating any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added by the polymerase, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals.

Description

FIELD [0001] This disclosure relates to an automated method for sequencing nucleic acids, such as DNA and RNA, which may be used for research and the diagnosis of disease in clinical applications. BACKGROUND [0002] Approaches to DNA sequencing over the past twenty years have varied widely. The use of enzymes and chemicals is making it possible to sequence the human genome. However, this effort takes enormous resources. [0003] Until recently, there were only two general sequencing methods available, the Maxam-Gilbert chemical degradation method (Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci., USA 74:560), and the Sanger dideoxy chain termination method (Sanger et at, 1977, Proc. Natl. Acad Sci., USA 74:5463). Using the dideoxy chain termination DNA sequencing method, DNA molecules of differing lengths are generated by enzymatic extension of a synthetic primer, using DNA polymerase and a mixture of deoxy- and dideoxy-nucleoside triphosphates. To perform this reaction, the DNA templat...

AI Technical Summary

Benefits of technology

"The present patent provides an improved method and device for sequencing nucleic acids by exposing them to an oligonucleotide primer and a polymerase in the presence of a mixture of nucleotides. The method allows several nucleic acids to be sequenced simultaneously at the molecular level. The method uses a donor and acceptor class of dyes that allow for multiplex sequencing, and the polymerase carries a fluorophore that emits a unique signal when excited. The unique signals are detected by a detector and converted into the nucleic acid sequence. The patent also describes attaching a polymerase to a substrate and adding the sample nucleic acid with anne. The technical effects of this patent include improved sequencing speed, accuracy, and sensitivity."

Problems solved by technology

However, this effort takes enormous resources.

However, because of the absence of a 3′-OH group on the ddNTP, phosphodiester bonds cannot be formed with the next incoming dNTPs.

This results in termination of the growing complementary DNA chain.

However, the methods disclosed in these patents still require the inconvenient step of separating the generated DNA fragments by size, using electrophoresis.

There are several disadvantages associated with using electrophoresis for nucleic acid sequencing.

Electrophoresis requires macroscopic separation, with the necessity of expensive reagents, long gel preparation time, tedious sample loading, the dangers of exposure to the neurotoxin acrylamide.

Macromolecular electrophoretic separation also exposes the technician to high voltage devices, requires prolonged electrophoresis time, produces gel artifacts, and requires calculations to adjust for dye mobilities.

Furthermore, sequencing runs only allow for the sequencing of less than 1000 bases at a time, which can be a substantial drawback to the sequencing of long stretches of the genome.

However, mass spectrometry devices are expensive, and because the method depends on size separation, it has a size resolution limit.

However, this method is limited by the separation process and requires very high detection sensitivity and wavelength selectivity due to the small sample size.

A disadvantage of macromolecular sequencing methods is that even though all of the DNA molecules start with identical nucleotides, they may quickly evolve into a mixed population.

One drawback to these methods, however, is that the DNA molecule which is being sequenced must be held in a stream, which often results in shearing of the DNA, especially at higher flow rates.

The sheared DNA molecule can not be accurately sequenced.

A drawback of these methods is that there is still a need for size separation (for example using electrophoresis) prior to determining the DNA sequence.

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