Method for determining allele frequencies

• US 7,078,168 B2
• Filed: 02/27/2002
• Issued: 07/18/2006
• Est. Priority Date: 02/27/2001
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Biotage AB (Biotage)
• Inventors: Sylvan, Anna
• Primary Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US10/085,774
• Publication Number:
  US 20030082566A1
• Time in Patent Office: 1,602 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 435/174, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present invention related to a method of determining the frequency of an allele in a population of nucleic acid molecules. The method comprises pooling the nucleic acid molecules of a population of nucleic acids, performing primer extension reactions using a primer which binds at a predetermined site located in nucleic acid molecules, and obtaining a pattern of nucleotide incorporation.

Claims

1. A method of determining the frequency of an allele in a population of nucleic acid molecules, said method comprising:

pooling the nucleic acid molecules of said population to provide a pooled nucleic acid sample;

performing primer extension reactions in a reaction mixture comprising said pooled nucleic acid sample and a primer which binds at a predetermined site located in said nucleic acid molecules, wherein said site is substantially adjacent to a polymorphic position of interest in said allele, to provide primer extension products, and wherein the primer extension reaction is performed by sequentially adding non-chain terminating nucleotides to the reaction mixture and quantitatively determining the incorporation or non-incorporation of each nucleotide as each nucleotide is added by bioluminometrically detecting the release of pyrophosphate;

obtaining a pattern of nucleotide incorporation in said primer extension products at the positions that correspond to said polymorphic position of interest; and

determining the frequency of said allele from said pattern of nucleotide incorporation.

2. The method according to claim 1 wherein ELIDA detection enzymes are used to detect the release of pyrophosphate.

3. The method according to claim 2 wherein a nucleotide-degrading enzyme is included during the primer extension reaction.

4. The method according to claim 1 wherein the nucleic acid molecules are immobilized on a solid support.

5. The method according to claim 1 wherein the amount or concentration of the nucleic acid in each sample of the population which is pooled, is determined prior to pooling.

6. The method according to claim 5 wherein the concentration of the nucleic acid in each sample of the population is determined by a primer-extension reaction prior to pooling.

7. The method according to claim 6 wherein the volume of each nucleic acid in each sample to be pooled is adjusted in view of the amount or concentration of nucleic acid present such that the pooled sample contains substantially the same amount or concentration of each nucleic acid molecule in the population.

8. The method according to claim 7 wherein a particular polymorphism is selected as a reference polymorphism and said primer extension reaction used to determine the concentration of nucleic acid in said sample is specific for said reference polymorphism.

9. The method according to claim 8 wherein said polymorphism is chosen such that it gives no background signals in a primer-extension reaction and that the signals are even.

10. The method according to claim 8 wherein said polymorphism is not present in a homopolymeric sequence and will not be preferentially amplified in any PCR-type reactions.

11. The method according to claim 8 wherein a reference sample containing said polymorphism is selected as the main reference from one of the homozygotes of one of the alleles of said polymorphism (Ref 1) and another reference (Ref 2) containing said polymorphism is selected from the other homozygote, and the reference samples are pooled and primer extension reactions are performed, and the pattern of nucleotide incorporation determined to determine the relative concentration of each reference sample.

12. The method according to claim 11 wherein the sample nucleic acid molecules to be tested are pooled individually with the reference samples.

13. A method of determining the amount of an allele in a sample of nucleic acid molecules, said method comprising:

performing primer extension reactions in a reaction mixture comprising said nucleic acid molecules, using a primer which binds at a predetermined site located in at least one said molecule wherein said site is substantially adjacent to a polymorphic position of interest in said allele, to provide primer extension products, and wherein the primer extension reaction is performed by sequentially adding non-chain terminating nucleotides to the reaction mixture and quantitatively determining the incorporation or non-incorporation of each nucleotide as each nucleotide is added by bioluminometrically detecting the release of pyrophosphate;

determining the type and number of nucleotides incorporated in said primer extension products at positions that correspond to the polymorphic position of interest, and determining the amount of occurrence of said allele in said sample by analyzing the type and number of nucleotides incorporated.

14. The method according to claim 13 wherein ELIDA detection enzymes are used to detect the release of pyrophosphate.

15. The method according to claim 14 wherein a nucleotide-degrading enzyme is included during the primer extension reaction.

16. The method according to claim 15 wherein the nucleic acid molecules are immobilized on a solid support.