Methods for genetic analysis of SARS virus
First Claim
1. An array comprising a plurality of nucleic acid probes, wherein said plurality of nucleic acid probes comprises each of the sequences listed in SEQ ID Nos. 1-238,192 wherein each different sequence is attached to the surface of the array in a different localized area.
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Abstract
The invention provides arrays and probes for resequencing a SARS virus using an array of probes that are complementary to a SARS reference sequence and to each possible single nucleotide substitution of the reference sequence. Methods of identifying mutations in viral sequences and methods of characterizing viral isolates are also provided. The invention also provides high throughput methods to monitor epidemics and pandemics caused by pathogens such as viruses.
21 Citations
29 Claims
- 1. An array comprising a plurality of nucleic acid probes, wherein said plurality of nucleic acid probes comprises each of the sequences listed in SEQ ID Nos. 1-238,192 wherein each different sequence is attached to the surface of the array in a different localized area.
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4. An array of nucleic acid probes immobilized on a solid support, the array comprising:
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(1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least ten nucleotides exactly complementary to a subsequence of a SARS virus reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the SARS virus reference sequence; and
(2) second, third and fourth probe sets, each probe set comprising a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to the corresponding probe from the first probe set or a subsequence of at least ten nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of monitoring an outbreak of a disease caused by a virus of interest comprising:
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isolating a nucleic acid sample from each of a plurality of individuals suspected of being infected with the virus of interest;
amplifying viral nucleic acid from the virus of interest in each of the nucleic acid samples;
hybridizing the amplified nucleic acids to a resequencing array comprising probes to a reference sequence of the virus of interest;
estimating at least part of the sequence of the virus of interest in each of the samples; and
comparing the sequences to determine the variation between individual isolates. - View Dependent Claims (22, 23, 24)
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25. An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes,
(1) a first probe set comprising a plurality of probes wherein each probe comprises a segment of at least ten nucleotides that is perfectly complementary to a subsequence of a first reference sequence, the subsequence including at least one interrogation position complementary to a corresponding nucleotide in the first reference sequence, (2) a second probe set comprising a plurality of probes wherein each probe comprises a subsequence of at least ten nucleotides that is perfectly complementary to a subsequence of a second reference sequence, the subsequence including at least one interrogation position complementary to a corresponding nucleotide in the second reference sequence, wherein the first reference sequence is a first isolate of a SARS virus and the second reference sequence is a second isolate of a SARS virus.
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28. A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising:
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(a) hybridizing a sample comprising the target nucleic acid to an array of nucleic acid probes immobilized on a solid support, the array comprising at least four sets of probes, (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least nine nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) second, third and fourth probe sets, each set comprising a probe for each interrogation position in the first probe set, each probe comprising a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least nine nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets;
provided the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U;
wherein the reference sequence is a SARS virus (b) comparing the relative specific binding of four corresponding probes from the first, second, third and fourth probe sets;
(c) assigning a nucleotide in the target sequence as the complement of the interrogation position of the probe having the greatest specific binding;
(d) repeating (b) and (c) by comparing the relative specific binding of a further four corresponding probes from the first, second, third and fourth probe sets until each nucleotide of interest in the target sequence has been assigned. - View Dependent Claims (29)
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Specification