System and method for temperature gradient capillary electrophoresis

• US 7,175,750 B2
• Filed: 11/05/2002
• Issued: 02/13/2007
• Est. Priority Date: 09/01/2000
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Spectrumedix LLC
• Inventors: Li, Qingbo, Liu, Zhaowei, Guo, Zhiyong
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US10/287,826
• Publication Number:
  US 20030138821A1
• Time in Patent Office: 1,561 Days
• Field of Search: 204451-458, 204/461, 204466-467
• US Class Current: 205/451
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Abstract

The present invention relates to a method for determining the presence of a mutation in a first sample comprising first nucleotides. The reference sample comprising reference nucleotides. The first sample and a reference sample are subjected to electrophoresis in the presence of at least one intercalating dye. During electrophoresis the temperature of the first sample and the reference sample is changed by an amount sufficient to change an electrophoretic mobility of at least one of the first or reference nucleotides. Fluorescence intensity data are obtained. The fluorescence intensity data are indicative of the presence of the first and reference nucleotides. At least one of the first sample or reference samples comprises products resulting from a polymerase chain reaction (PCR), the products not having been desalted prior to electrophoresis.

Claims

1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample, comprising:

providing an unknown double-stranded polynucleotide sample;

subjecting the unknown double-stranded polynucleotide sample to temperature gradient gel electrophoresis, the unknown double-stranded polynucleotide sample including non-desalted polymerase chain reaction (PCR) products;

subjecting a reference double-stranded polynucleotide to temperature gradient electrophoresis;

irradiating the unknown double-stranded polynucleotide with light to generate a first spectroscopic signal,

irradiating the reference double-stranded polynucleotide with light to generate a second spectroscopic signal;

converting the first and second spectroscopic signals into first and second data; and

determining the presence of a single nucleotide polymorphism or a mutation in the unknown double-stranded polynucleotide sample based on the first and second data.

2. The method of claim 1, wherein the spectroscopic signals are fluorescence signals.

3. The method of claim 1, wherein the spectroscopic signals are absorbance signals.

4. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample, comprising:

determining a first parameter based on a first spectroscopic signal resulting from irradiating an unknown double-stranded polynucleotide sample with light the unknown double-stranded polynucleotide sample having been subjected to temperature gradient gel electrophoresis in the presence of non-desalted polymerase chain reaction (PCR) products;

determining a second parameter based on a second spectroscopic signal resulting from irradiating a reference double-stranded polynucleotide sample with light, the reference double-stranded polynucleotide sample having been subjected to temperature gradient electrophoresis; and

comparing the first and second parameters to determine whether there is a single nucleotide polymorphism or a mutation in the unknown double-stranded polynucleotide sample.

5. The method of claim 4, wherein the spectroscopic signals are fluorescence signals.

6. The method of claim 4, wherein the spectroscopic signals are absorbance signals.

7. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample having single-stranded nucleic acid PCR products, comprising:

providing an unknown double-stranded polynucleotide sample;

subjecting the unknown double-stranded polynucleotide sample to temperature gradient electrophoresis in the presence of single-stranded DNA associated with PCR amplification of double-stranded polynucleotides of the unknown double-stranded polynucleotide sample;

subjecting a reference double-stranded polynucleotide sample to temperature gradient electrophoresis;

irradiating the unknown double-stranded polynucleotide sample with light to generate a first spectroscopic signal, and irradiating the reference double-stranded polynucleotide sample with light to generate a second spectroscopic signal;

converting the first and second spectroscopic signals into first and second data; and

determining the presence of a single nucleotide polymorphism or a mutation in the unknown double-stranded polynucleotide sample based on the first and second data.

8. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample having single-stranded nucleic acid PCR products, comprising:

determining a first parameter based on a first spectroscopic time signal resulting from irradiating an unknown double-stranded polynucleotide sample with light, the unknown double-stranded polynucleotide sample having been subjected to temperature gradient electrophoresis in the presence of single-stranded DNA associated with PCR amplification of double-stranded polynucleotides of the unknown double-stranded polynucleotide sample;

determining a second parameter based on a second spectroscopic time signal resulting from irradiating a reference double-stranded polynucleotide with light that has been subjected to temperature gradient electrophoresis; and

comparing the first and second parameters to determine the presence of a single nucleotide polymorphism or a mutation in the unknown double-stranded polynucleotide.

9. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second different unknown double-stranded polynucleotide samples, each of the samples comprising first and second member double-stranded polynucleotides, the members of the first sample and the members of the second sample being different sizes, comprising:

while subjecting the first and second samples to electrophoresis within the same capillary in the presence of one another, changing the temperature at a first non-zero rate for a first period of time by an amount sufficient to change an electrophoretic mobility of the first member of the first unknown sample relative to an electrophoretic mobility of the second member of the first unknown sample and then changing the temperature at a second, different non-zero rate for a second period of time by an amount sufficient to change an electrophoretic mobility of the first member of the second unknown sample relative an electrophoretic mobility of the second member of the second unknown sample;

irradiating the members of the first unknown sample with light to generate a first spectroscopic signal, and irradiating the members of the second unknown sample with light to generate a second spectroscopic signal; and

determining the presence of a single nucleotide polymorphism or a mutation in the first unknown sample based on the first spectroscopic signal and determining the presence of a single nucleotide polymorphism or a mutation in the second unknown sample based on the second spectroscopic signal.

10. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second different unknown double-stranded polynucleotide samples, member double-stranded polynucleotides of the first unknown sample being a different size from member double-stranded polynucleotides of the second unknown sample, comprising:

determining a first parameter from a first spectroscopic signal obtained by irradiating the first unknown double-stranded polynucleotide sample with light;

determining a second parameter from a second spectroscopic signal obtained by irradiating the second unknown double-stranded polynucleotide sample with light

wherein, prior to irradiating the first and second unknown polynucleotide samples with light, the first and second unknown polynucleotide samples were subjected to temperature gradient electrophoresis together along the sample capillary, wherein subjecting the first and second unknown double-stranded polynucleotide samples to electrophoresis comprised changing the temperature at a first non-zero rate for a first portion of the time and changing a temperature at a second, different and non-zero rate for a second portion of the time during the electrophoresis;

comparing each of the first and second parameters with a reference parameter to determine whether there is a single nucleotide polymorphism or a mutation in either of the first or second unknown double-stranded polynucleotide samples.

11. A capillary-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second unknown double-stranded polynucleotides, member polynucleotides of the first unknown double-stranded polynucleotide sample being smaller than member polynucleotides of the second unknown double-stranded polynucleotide sample, the method comprising:

subjecting the first and second unknown double-stranded polynucleotide samples to electrophoresis together in the sample capillary;

during electrophoresis, changing the temperature of a central portion of the capillary from a first temperature T₁ to a second different temperature T₂;

irradiating the first unknown double-stranded polynucleotide sample with light to generate a first spectroscopic signal, and irradiating the second unknown double-stranded polynucleotide sample with light to generate a second spectroscopic signal;

converting the first and second pairs of spectroscopic signals into first and second data; and

determining the presence of a single nucleotide polymorphism or mutation in the first unknown double-stranded polynucleotide sample based on the first data and determining the presence of a single nucleotide polymorphism or mutation in the second unknown double-stranded polynucleotide sample based on the second data.

12. In a method for detecting mutations in a polynucleotide sample by subjecting the polynucleotides to temperature gradient electrophoresis and obtaining spectroscopic intensity data indicative of the presence of the polynucleotides, the improvement comprising:

thermally-contacting the sample polynucleotides with a chilled gas by contacting a capillary in which the polynucleotides are migrating with the chilled gas to reduce the temperature of the polynucleotides after the polynucleotides have been subjected to temperature gradient electrophoresis and prior to obtaining the spectroscopic intensity data.

13. The method according to claim 12, wherein the polynucleotides comprise homoduplex or heteroduplex DNA fragments.

14. The method of claim 12, wherein the temperature of the chilled gas has a temperature of less than about 15°

C.

15. The method of claim 12, wherein actively reducing the temperature comprises thermoelectrically cooling the polynucleotides.

16. The method of claim 12, wherein actively reducing the temperature comprises thermally contacting the nucleotides with a chilled fluid.

17. The method according to claim 12, wherein, when the fluorescence intensity data are obtained, the temperature of the polynucleotides is less than about 30°

C.