Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides
First Claim
1. A statistically-based random-priming method for determining nucleotide sequence in a nucleic acid template having a completely unknown or partly known nucleotide sequence by priming within a region of the template for which the nucleotide sequence is not known, the method comprising the steps of:
- a) supplying a template for which the approximate total length of unknown nucleotide sequence is known;
b) selecting a primer or primer combination whose length or lengths relative to the template length are such that the probability P(1) of priming at a single site within the total length of unknown nucleotide sequence in the template is about 0.291-0.368, but excluding any primer that would prime in any part of the template where the nucleotide sequence is known;
c) forming an incubation mixture comprising;
i) the template;
ii) the primer or primer combination selected in step b); and
iii) a polymerizing enzyme;
d) incubating the mixture of step c) under conditions appropriate for primed synthesis of DNA to generate products suitable for determining the nucleotide sequence in the template;
e) analyzing the products of step d) to determine nucleotide sequence, which will be determinable only if priming occurred in step d) and was at a single site in the template; and
f) if necessary, repeating steps b)-e), using different primers or primer combinations, until the nucleotide sequence has been determined.
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Abstract
Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA.
Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.
154 Citations
18 Claims
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1. A statistically-based random-priming method for determining nucleotide sequence in a nucleic acid template having a completely unknown or partly known nucleotide sequence by priming within a region of the template for which the nucleotide sequence is not known, the method comprising the steps of:
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a) supplying a template for which the approximate total length of unknown nucleotide sequence is known; b) selecting a primer or primer combination whose length or lengths relative to the template length are such that the probability P(1) of priming at a single site within the total length of unknown nucleotide sequence in the template is about 0.291-0.368, but excluding any primer that would prime in any part of the template where the nucleotide sequence is known; c) forming an incubation mixture comprising; i) the template; ii) the primer or primer combination selected in step b); and iii) a polymerizing enzyme; d) incubating the mixture of step c) under conditions appropriate for primed synthesis of DNA to generate products suitable for determining the nucleotide sequence in the template; e) analyzing the products of step d) to determine nucleotide sequence, which will be determinable only if priming occurred in step d) and was at a single site in the template; and f) if necessary, repeating steps b)-e), using different primers or primer combinations, until the nucleotide sequence has been determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A statistically-based directed-priming method that uses primers selected from a primer library to determine nucleotide sequence in a nucleic acid template having a nucleotide sequence which is partly known and partly unknown, the method comprising the steps of:
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a) supplying a template of known approximate length; b) supplying a primer library containing primers having lengths relative to the template length such that the probability P(O) that an individual primer will have no perfectly complementary priming site in any unknown nucleotide sequence in the template is greater than about 0.25, said library being of a size that the average priming interval is less than about half the average length of nucleotide sequence that can be determined from a single priming site; c) selecting from the primer library a primer that is perfectly complementary to one and only one site in the known nucleotide sequence in the template; d) forming an incubation mixture comprising; i) the template; ii) the primer selected in step c); and iii) a polymerizing enzyme; e) incubating the mixture of step d) under conditions appropriate for primed synthesis of DNA to generate products suitable for determining nucleotide sequence in the template; and f) analyzing the products of step e) to determine nucleotide sequence, which will be determinable only if the priming step e) was at a single site in the template. - View Dependent Claims (10, 11, 12)
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13. A statistically-based combined random- and directed-priming method for determining nucleotide sequence in a nucleic acid template having a completely unknown or partly known nucleotide sequence, comprising the steps of:
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a) supplying a template of known approximate length and for which the approximate total length of unknown nucleotide sequence is known; b) supplying a primer library containing primers having lengths relative to the template length such that the probability P(0) that an individual primer will have no perfectly complementary priming site in any unknown nucleotide sequence part in the template is greater than about 0.25, said library being of a size that the average priming interval is less than about half the average length of nucleotide sequence that can be determined from a single priming site; c) selecting from the primer library a primer or primer combination whose length or lengths are such that the probability P(1) of priming at a single site within the total length of the unknown nucleotide sequence part in the template is about 0.291-0.368, but excluding any primer that would prime in any part of the template for which the nucleotide sequence is known; d) forming an incubation mixture comprising; i) the template; ii) the primer or primer combination selected in step c); and iii) a polymerizing enzyme; e) incubating the mixture of step d) under conditions appropriate for primed synthesis of DNA to generate products suitable for determining nucleotide sequence in the template; f) analyzing the products of step e) to determine nucleotide sequence, which will be determinable only if priming occurred in step e) and was at a single site in the template; g) if necessary, repeating steps c)-f), using different primers or primer combinations, until nucleotide sequence information is determined; h) selecting from the primer library a primer that is perfectly complementary to one and only one site in the nucleotide sequence which is known in the template; i) forming an incubation mixture comprising; i) the template; ii) the primer selected in step h; and iii) a polymerizing enzyme; j) incubating the mixture of step i) under conditions appropriate for primed synthesis of DNA to generate products suitable for determining nucleotide sequence in the template; and k) analyzing the products of step j) to determine nucleotide sequence, which will be determinable only if the priming in step j) was at a single site in the template. - View Dependent Claims (14, 15, 16, 17, 18)
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Specification