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US 2006/0051801 A1Published application
Representational fragment amplification
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorGregory Sgarlato
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Abstract
Methods and kits are provided for non-preferential amplification of a population of nucleic acids. The methods allow for a high degree of amplification, where representation in the final population has a direct linear relationship with the starting material. The amplification product is useful as a probe for hybridization; for generation of libraries, for sequencing, and the like.
Claims7
A method for non-preferential replication of a starting pool of nucleic acids, the method comprising:
- synthesizing cDNA from said starting pool, utilizing an oligo dT primer comprising restriction sites at least one frequent cutter restriction enzyme;
- synthesizing a DNA strand complementary to said cDNA to produce double stranded DNA;
- ligating linkers comprising restriction sites for said frequent cutter restriction enzyme to the 5′ end of said double stranded DNA;
- digesting the product of said ligating step with said frequent cutter restriction enzyme;
- ligating non-phosphorylated linkers to fragments of said digesting step;
- amplifying the product of said ligating step with a single primer;
- wherein the product of said amplifying step is non-preferentially amplified about 103-fold from said starting pool.
4 more elements
A kit for non-preferential amplification of a starting pool of nucleic acids, the kit comprising:
- an oligo dT primer comprising restriction sites at least one frequent cutter restriction enzyme;
- linkers comprising restriction sites for said frequent cutter restriction enzyme to the 5′ end of said double stranded DNA;
- and
- instructions for use.
1 more element
Dependencies are inferred from the claim text.