Polynucleotide sequencing method

Details Polynucleotide sequencing method Polynucleotide sequencing method

1999-02-16

2003-10-21

Whisenant, Ethan (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S026200

Reexamination Certificate

active

06635419

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for sequencing polynucleotides. In particular, the invention relates to an improved terminator sequencing method that expands the sequenceable range of bases adjacent to sequencing primers.
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BACKGROUND OF THE INVENTION
Sequence determination of polynucleotides is playing an increasingly important role in biochemical research and industry. For example, whole genomic sequences have been reported recently for the eukaryotes,
C. elegans
and
S. Cerevisiae
(e.g., see
Science
282:2012 (1998)), and substantial efforts are currently underway to sequence the human genome. Knowledge of biological polynucleotide sequences is useful not only to characterize and compare gene sequences and associated gene products from different organisms, but also for the development of diagnostic tests, e.g., for detecting genetic mutations, determining genotypes, diagnosing disease, forensics, and identifying therapeutic targets.
Over the last two decades, there have been two principal approaches to nucleotide sequence determination: the chain termination method of Sanger and Coulson (1977), and the chemical degradation method of Maxam and Gilbert (1977). Both methods require the generation of one or more sets of labeled DNA fragments, each having a common origin and each terminating with a known base. The set or sets of fragments must then be separated by size to reveal the target sequence.
More recently, the chain termination method has become the standard method for all currently available automated DNA sequencing machines. This method generally involves enzymatic extension of a 5′-primer along a target template strand in the presence of the four standard deoxyribonucleotide bases, plus one or more dideoxynucleotide terminators. Incorporation of a terminator during primer extension results in a mixture of products of variable length, each terminating at its 3′-end with the terminator. As originally proposed, four separate sequencing reactions were performed for a given target sequence, one for each dideoxynucleotide base-type. The products from each mixture were then resolved in four separate lanes on the basis of size, and the target sequence was determined from the relative order of migration of the fragments.
Modifications to the Sanger method were subsequently developed which use spectrally distinguishable fluorescent labels attached to either the 5′-extension primers (Smith, 1986) or the 3′-dideoxy terminator bases (Prober, 1987; Prober 1995; Bergot, 1991). In the former case, extension reactions must be performed separately for each different terminator base in the presence of a suitably labeled primer (a different label for each different terminator), after which the labeled extension products can be combined and resolved in a single separation path. In the latter case, primer extension can be performed in a single reaction mixture, since base-specific label incorporation is performed with high fidelity by a polymerase enzyme, followed by separation in a single separation lane.
The labeled terminator method has become the sequencing method of choice because it involves significantly fewer reaction vessels and is more convenient to use. However, this method has been limited due to difficulty in routinely identifying sequencing fragment nucleotides close to the extension primers. The extension reactions require high concentrations of labeled terminators to ensure efficient production of sequencing fragments. Unfortunately, residual terminator bases or their breakdown products comigrate with short sequencing fragments because they have similar mobility properties. As a consequence, sequencing data for short sequencing fragments has been either unreliable or entirely unusable. Theoretically, it is possible to remove residual terminator bases prior to sequence analysis, e.g., by a preliminary size-based separation step. However, this introduces an extra step to the sequencing process that encumbers large-scale sequence analysis. Moreover, such preliminary purification may have to be repeated before enough residual terminator material has been removed.
Accordingly, it is an object of the present invention to provide a labeled terminator sequencing method that permits sequence determinations close to the extension primer.
A further object of the invention is to overcome the contamination problems that have been associated with previous labeled terminator sequencing methods.
SUMMARY OF THE INVENTION
The present invention provides, in one aspect, a method for determining a sequence of one or more nucleotides in a target polynucleoti

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