Method for genome complexity reduction and polymorphism detection
AssigneeUniversity of Florida Research Foundation, Inc.
InventorsMatias Kirst, Marcio Fernando Ribeiro De Resende, Jr., Leandro Gomide Neves, Christopher Dervinis, Kelly Mayrink Balmant
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Abstract
The present invention provides methods to produce a reduced representation of a genome for sequencing and DNA polymorphism detection. In particular, the invention provides PCR-based methods, with normalization of the amplified products using a duplex-specific nuclease, in order to reduce over-representation of PCR products. Oligonucleotides for use in the disclosed method are also provided.
Claims36
A method for producing a reduced representation of a genome, comprising the steps of:
- (a) amplifying by polymerase chain reaction (PCR) regions of the genome using a first oligonucleotide primer set to produce a first nucleic acid product;
- (b) normalizing the nucleic acid product of step (a) by contacting it with a duplex-specific nuclease for an effective amount of time to digest the most abundant double-stranded nucleic acid product of step (a);
- and
- (c) amplifying by PCR the normalized nucleic acid product of step (b) using a second oligonucleotide primer set to produce a second nucleic acid product having a linker sequence, wherein a primer of the second oligonucleotide primer set comprises said linker sequence;
- and
- wherein the nucleic acid product of step (c) represents a reduced representation of the genome.
A method for producing a reduced representation of a genome, comprising the steps of:
- (a) amplifying by polymerase chain reaction (PCR) regions of said genome using a single oligonucleotide primer set, wherein a primer of the oligonucleotide primer set comprises a linker sequence, and said oligonucleotide primer set results in the addition of said linker sequence;
- and
- (b) normalizing the nucleic acid product of step (a) by contacting it with a duplex-specific nuclease for an effective amount of time to digest the most abundant double-stranded nucleic acid of step (a);
- wherein the nucleic acid product of said method represents a reduced representation of said genome.
Dependencies are inferred from the claim text.