US 5,972,693 A · Apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing

Abstract

This invention provides methods by which biologically derived DNA sequences in a mixed sample or in an arrayed single sequence clone can be determined and classified without sequencing. The methods make use of information on the presence of carefully chosen target subsequences, typically of length from 4 to 8 base pairs, and preferably the length between target subsequences in a sample DNA sequence together with DNA sequence databases containing lists of sequences likely to be present in the sample to determine a sample sequence. The preferred method uses restriction endonucleases to recognize target subsequences and cut the sample sequence. Then carefully chosen recognition moieties are ligated to the cut fragments, the fragments amplified, and the experimental observation made. Polymerase chain reaction (PCR) is the preferred method of amplification. Several alternative embodiments are described which capable of increased discrimination and which use TypeIIS restriction endonucleases, various capture moieties, or samples of specially synthesized cDNA. Another embodiment of the invention uses information on the presence or absence of carefully chosen target subsequences in a single sequence clone together with DNA sequence databases to determine the clone sequence. Computer implemented methods are provided to analyze the experimental results and to determine the sample sequences in question and to carefully choose target subsequences in order that experiments yield a maximum amount of information.

Claims38

Dependent claims

An apparatus programmed for analyzing signals produced from one or more samples comprising a plurality of nucleic acids, said apparatus comprising one or more computer systems programmed for:

(a) inputting one or more of said signals, each said input signal being produced from a nucleic acid in said samples and comprising a representation of (i) the length between occurrences of target nucleotide subsequences in said nucleic acid of said samples, said target nucleotide subsequences comprising predetermined nucleotide sequences recognized by recognition means, and (ii) the identities of said target nucleotide subsequences in said nucleic acid, or the identities of sets of target nucleotide subsequences which have members including said target nucleotide subsequences in said nucleic acid;
(b) searching a nucleotide sequence stored in a nucleotide sequence database to determine both (i) if said target nucleotide subsequences occur in said nucleotide sequence, and (ii) the lengths in nucleotides between any such occurrences, said nucleotide sequence database comprising a plurality of known nucleotide sequences that may be present in said samples;
and
(c) predicting that a nucleic acid having a nucleotide sequence stored in said nucleotide sequence database produces one of said input signals if said nucleotide sequence is determined by said searching to have both (i) the same length between occurrences of target nucleotide subsequences as is represented by said one of said input signals, and (ii) the same target nucleotide subsequences as are represented by said one of said input signals, or target nucleotide subsequences that are members of the sets of target nucleotide subsequences as are represented by said one of said input signals.

1 more element

An apparatus programmed with the means for analyzing signals produced from one or more samples comprising a plurality of nucleic acids, comprising one or more computer systems programmed for:

(a) selecting target nucleotide subsequences or sets of target nucleotide subsequences, wherein said target nucleotide subsequences comprise predetermined subsequences recognized by recognition means;
(b) inputting one or more signals, each said input signal being produced from a nucleic acid in said samples and comprising a representation of (i) the length between occurrences of said subsequences in said nucleic acid of said samples, and (ii) the identities of said target nucleotide subsequences in said nucleic acid, or the identities of said sets of target nucleotide subsequences in said nucleic acid;
(c) predicting nucleotide sequences stored in a nucleotide sequence database that can produce said input signals, wherein said predicting is by a method comprising searching said nucleotide sequences for occurrences of said target nucleotide subsequences and for the length between occurrences of said target subsequences, wherein a nucleotide sequence from said database is predicted to produce a particular input signal when said nucleotide sequence has occurrences of the same target nucleotide subsequence and the same length between occurrences of target nucleotide subsequences as are represented by the input signal, and wherein said nucleotide sequence database comprises a plurality of known nucleotide sequences that may be present in said samples;
(d) outputting indications of said input signals and of said predicted nucleotide sequences stored in said database;
and
(e) controlling said selecting to select target nucleotide subsequences or sets of target nucleotide sequences, controlling said inputting to input said produced signals, controlling said predicting to predict nucleotide sequences in said database that can produce said input signals, and controlling said outputting to output indications of said input signals and said sequences in said database that are predicted to produce said input signals, each said controlling being performed in a manner responsive to user requests.

3 more elements

An apparatus programmed for selecting target nucleotide subsequences comprising one or more computer systems programmed for:

(a) choosing target nucleotide subsequences or sets of target nucleotide subsequences, wherein criteria for said choosing are determined according to an optimization method;
(b) determining a pattern of signals that are predicted to be produced by said chosen target nucleotide subsequences or said chosen sets of target nucleotide subsequences, said determining being done by a method comprising searching nucleotide sequences stored in a nucleotide sequence database both for occurrences of said chosen target nucleotide subsequences or occurrences of target nucleotide subsequences that are members of said chosen sets of target nucleotide subsequences and for the length in nucleotides between such occurrences and storing said determined occurrences and said determined lengths in said pattern, wherein said database comprises a plurality of known nucleotide sequences, and wherein said signals comprise (i) the length between said occurrences of said chosen target nucleotide subsequences in a nucleotide sequence in said nucleotide sequence database, and (ii) the identities of said chosen target nucleotide subsequences that occur in said sequence in said database, or the identities of target nucleotide subsequences that are members of said chosen sets of target nucleotide subsequences that occur in said nucleotide sequence in said nucleotide sequence database;
(c) ascertaining an amount of information in said determined pattern of predicted signals, said amount being indicated by the numerical value of an information measure, said information measure being computed from said determined pattern of predicted signals;
(d) controlling according to said optimization method the repetitive performance of said choosing, said determining, and said ascertaining in order to obtain a plurality of said values of said information measure;
and
(e) selecting those target nucleotide subsequences or sets of target nucleotide subsequences that maximize the amount of information in said pattern of predicted signals as indicated by said values of said information measure.

3 more elements

Dependencies are inferred from the claim text.

Apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing

Life of this patent

Apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing

Life of this patent

Abstract

Claims38