Strategies for high throughput identification and detection of polymorphisms

• US 10,095,832 B2
• Filed: 01/05/2018
• Issued: 10/09/2018
• Est. Priority Date: 06/23/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Van Der Poel, Henricus Johannes Adam
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/862,956
• Publication Number:
  US 20180137241A1
• Time in Patent Office: 277 Days
• Field of Search: 435 912
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2539/107 Representational Difference Analysis [RDA]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 30/10 by measuring physical properties, e.g. mass
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

The invention relates to a method for identifying one or more polymorphisms in nucleic acid samples, comprising: (a) performing a reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein the reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using one or more primers to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence to indicate origin of each library obtained by the reproducible complexity reduction; (b) combining the plurality of libraries to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences; (c) aligning the sequences to obtain an alignment; and (d) identifying one or more polymorphisms in the plurality of nucleic acid samples.

Claims

1. A kit for use in a method for detecting one or more polymorphisms in a plurality of nucleic acid samples, comprising:

(a) an adaptor comprising a 3′

-T overhang and (b) a set of primers for PCR amplification, wherein at least one of the adaptor and the primers comprises a sample-specific identifier sequence capable of indicating sample origin of an amplification product of the plurality of nucleic acid samples.

2. The kit according to claim 1, wherein the adaptor comprises the sample-specific identifier sequence.

3. The kit according to claim 1, wherein at least one of the primers comprises the sample-specific identifier sequence.

4. The kit according to claim 1, wherein the adaptor comprises a PCR primer-binding sequence for hybridizing to a PCR primer.

5. The kit according to claim 1, wherein the adaptor comprises a sequencing primer-binding sequence for hybridizing to a sequencing primer.

6. The kit according to claim 1, wherein the adaptor comprises sequences complementary to sequences attached to a solid support for annealing the adaptor to the solid support.

7. The kit according to claim 1, wherein the adaptor comprises sequences complementary to sequences attached to a bead for annealing the adaptor to the bead.

8. The kit according to claim 1, wherein at least one of the primers is phosphorylated.

9. The kit according to claim 1, wherein at least one of the primers comprises sequences at its 3′

-end for hybridizing to a subset of nucleic acid sample fragments.

10. The kit according to claim 1, wherein the kit further comprises a biotinylated capture oligonucleotide hybridization probe for capturing a subset of nucleic acid sample fragments.

11. The kit according to claim 1, wherein the kit further comprises a polymerase for a polymerase chain reaction (PCR).

12. The kit according to claim 11, wherein the polymerase substantially lacks 3′

-5′

exonuclease activity.