Nucleic acid sequencing with simultaneous quantitation

• US 7,655,443 B1
• Filed: 05/05/2000
• Issued: 02/02/2010
• Est. Priority Date: 05/07/1999
• Status: Expired due to Fees

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Yager, Thomas D, August, M Jason
• Primary Examiner(s): Sisson; Bradley L
• Application Number:
  US09/565,249
• Time in Patent Office: 3,560 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 435/810, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/24.31
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 2545/101 with an internal standard/control

Abstract

Simultaneous sequencing and quantitation of a nucleic acid analyte in a sample using the same reagents for both assays is achieved by processing a sample containing, or suspected of containing the nucleic acid analyte of interest using a single set of reagents through a plurality of thermocycles to obtain a mixture of labeled polynucleotides which are used for the determination of both sequence information about the target nucleic acid and the amount of target nucleic acid present in the sample. The fragments are separated on the basis of size, for example by electrophoresis, and the label associated with the separated fragments is detected. The positions of the separated nucleic acid fragments are evaluated to obtain information about the sequence of the target nucleic acid analyte, and the intensity of a signal derived from the label associated with one or more of the separated fragments is evaluated to determine the quantity of the target nucleic acid analyte in the sample. Only one label is needed for both sequencing and quantitation, although two or more labels may be used if bidirectional sequencing is concurrently performed.

Claims

1. A method for sequencing and quantitation of a target nucleic acid analyte in a sample, comprising the steps of:

(a) generating a set of nucleic acid sequencing fragments by processing one or more aliquots of the sample using a set of reagents containing a set of forward and reverse sequencing primers that flank a region of interest of the target nucleic acid between the primers, wherein the primers are sufficiently complementary to the respective 5′

ends of the sense and anti-sense strands of the target nucleic acid to produce through a plurality of thermocycles a mixture of primer extension nucleic acid sequencing fragments terminating at each of a particular base type within the target nucleic acid and incorporating a detectable label indicative of the terminating base type that is capable of detection to determine the sequence of the nucleic acid and to determine the amount of target nucleic acid present in the sample, said processing being carried out within a quantitative regime wherein the plurality of thermocycles is performed at cycle numbers where a plot of log of the amount of product as a function of cycle number is linear;

(b) determining the sequence of the target nucleic acid analyte by separating the labeled nucleic acid sequencing fragments in the modified sample in order of size and detecting the order of separation of the labels associated with the separated sequencing fragments generated in (a); and

(c) determining the quantity of the target nucleic acid analyte present in the sample by correlating the intensity of a signal obtained from the label of one or more of the sequencing fragments generated in (a) with the amount of the target nucleic acid analyte present in the sample, using a reference standard,

wherein the sequencing fragments used to determine the sequence of the target nucleic acid and the sequencing fragments used to determine the quantity of target polynucleotide are produced using primers having the same nucleic acid sequence.

2. The method of claim 1, wherein the label of the terminated sequencing fragments is detected and quantitated for the determination of both sequence information about the target nucleic acid and the amount of target nucleic acid present in the sample.

3. The method of claim 1, wherein a labeled full-length primer extension product is detected and quantitated for determination of the amount of target nucleic acid present in the sample.

4. The method of claim 3, wherein the sample is processed in two parallel replicate reactions to produce both full-length product and terminated polynucleotide sequencing fragments, said two reactions including a first reaction which is stopped after a first number of thermal cycles such that the production of full-length product is within a quantitative regime, and a second reaction which is stopped after a second number of thermal cycles greater than the first number of thermal cycles such that the production of sequencing fragments is within a quantitative regime.

5. The method of claim 3, wherein the sample is processed in a single reaction and a first aliquot of the single reaction is removed after a first number of thermal cycles such that the production of full-length product is within a quantitative regime, said full length-product in the first aliquot being used for determination of the amount of target nucleic acid in the sample, and wherein the sample is further processed after the removal of the first aliquot for an additional number of thermal cycles such that production of sequencing fragments is within a quantitative regime.

6. The method of claim 3, wherein an internal polynucleotide reference standard is added to the sample in a known amount and coprocessed in the same reaction vessel to produce a detectable reference product, and wherein the intensity of a signal derived from the reference product is compared to the intensity of the signal derived from the label associated with the separated fragments to determine the quantity of the target nucleic acid analyte in the sample.

7. The method of claim 6, wherein the target nucleic acid is an RNA target, and wherein the method further comprises the step of reverse transcribing the RNA target to produce a cDNA template prior to step (a).

8. The method of claim 1, wherein an internal polynucleotide reference standard is added to the sample in a known amount and coprocessed in the same reaction vessel to produce a detectable reference product, and wherein the intensity of a signal derived from the reference product is compared to the intensity of the signal derived from the label associated with the separated fragments to determine the quantity of the target nucleic acid analyte in the sample.

9. The method of claim 8, wherein the target nucleic acid is an RNA target, and wherein the method further comprises the step of reverse transcribing the RNA target to produce a cDNA template prior to step (a).

10. The method of claim 8, wherein the label of the terminated sequencing fragments is detected and quantitated for the determination of both sequence information about the target nucleic acid and the amount of target nucleic acid present in the sample.

11. The method of claim 1, wherein both a labeled full-length product and labeled terminated sequencing fragments are detected and quantitated for the determination of the amount of target nucleic acid present in the sample.

12. The method of claim 1, wherein the target nucleic acid is an RNA target, and wherein the method further comprises the step of reverse transcribing the RNA target to produce a cDNA template prior to step (a).

13. The method of claim 12, wherein the target nucleic acid analyte is HIV-1, and wherein the set of reagents includes primers flanking a region of interest within the HIV-1 genome.

14. The method of claim 13, wherein an internal polynucleotide reference standard is added to the sample in a known amount and coprocessed in the same reaction vessel to produce a detectable reference product, and wherein the intensity of a signal derived from the reference product is compared to the intensity of the signal derived from the label associated with the separated fragments to determine the quantity of the target nucleic acid analyte in the sample.