Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-20
2003-10-14
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091520, C435S091510, C536S024300, C536S024200
Reexamination Certificate
active
06632611
ABSTRACT:
FIELD OF INVENTION
The present invention is in the field of nucleic acid hybridization. More specifically, the present invention is in the field of nucleic acid hybridization and enrichment.
BACKGROUND OF THE INVENTION
Methods of amplifying target DNA sequences can be costly and complicated, often requiring large numbers of specific primer sequences that must be synthesized for each experiment. One aspect of the present invention provides a process that is cost effective and straight forward for enriching and amplifying specific nucleic acid sequences including polymorphic regions, chromosomal regions and whole chromosomes.
SUMMARY OF INVENTION
The currently claimed invention provides novel methods for the enrichment and amplification of target sequences from a nucleic acid population using selection probes. The invention further provides for analysis of the sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism.
In a first embodiment, the currently claimed invention provides a method of amplifying a population of target sequences wherein each of said target sequences comprises a region of interest. The method comprises fragmenting a sample nucleic acid population, ligating an adaptor sequence to the 5′ end of the fragments and exposing the fragments to selection probes. The selection probes comprise a region that is complementary to a region of interest and a unique sequence at the 5′ end. Target sequences and selection probes are allowed to hybridize forming probe-target complexes. The target sequence is then extended at the 3′ end using the selection probe as a template. As a result the extended target will contain a unique sequence at its 3′ end that is the complement of the unique sequence at the 5′ end of the selection probe. The target can then be selectively amplified using a first primer that recognizes the unique sequence added to the 3′ end and a second primer that recognizes the 5′ adaptor sequence.
In another embodiment the presently claimed invention provides a method of amplifying a population of target sequences wherein each of said target sequences comprises a region of interest. The method comprises fragmenting a double stranded population, making the population single stranded by selectively degrading one strand, exposing the fragments to selection probes to form target-probe complexes, removing single stranded nucleic acids, leaving completely double stranded probe-target complexes, ligating adaptors to the ends of the probe-target complexes and amplifying the probe target complexes using primers that are complementary to the adaptors. The selection probes comprise a region that is complementary to the region of interest.
In yet another embodiment, the invention relates to a kit comprising reagents and instructions for reproducibly amplifying a population of target sequences. The kit may comprise a plurality of selection probes, reagents and instructions necessary for amplification of one or more regions of interest. The kit may also comprise a means to generate selection probes.
The present invention also provides methods for genotyping an individual which may further comprise contacting the amplified target sequences with a solid support comprising nucleic acid probes, and detecting the presence or absence of hybridization of the target sequence to the nucleic acid probes on the solid support. The immobilized probes in a preferred embodiment are capable of interrogating one or more polymorphic sites. The identity of the polymorphic base is determined from the hybridization information. In a preferred embodiment, the solid support, which may comprise nucleic acid probes, can be selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
In another embodiment the presently claimed invention provides a method for making a population of selection probes to be used in the current invention. The selection probes are particularly useful if they are a subset of a complex population. For example a particularly useful population of selection probes would be a subset of a genome.
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Dong Shoulian
Su Xing
Affymetrix Inc.
McGarrigle Philip L.
Tung Joyce
Wells Sandra E.
Whisenant Ethan
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