System and method for determining the presence of methylated cytosines in polynucleotides

• US 20040048276A1
• Filed: 04/11/2003
• Published: 03/11/2004
• Est. Priority Date: 10/11/2000
• Status: Abandoned Application

• Current Assignee: Spectrumedix LLC
• Original Assignee: Spectrumedix LLC
• Inventors: Li, Qingbo, Liu, Zhaowei
• Application Number:
  US10/411,103
• Publication Number:
  US 20040048276A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

The present invention relates to a method for determining the presence of a methylated cytosine in a first sample comprising a first nucleotide containing compound. The first sample and a reference NCC are subjected to electrophoresis in the presence of at least one intercalating dye. During electrophoresis the temperature of the first NCC and the reference NCC is changed by an amount sufficient to change an electrophoretic mobility of at least one of the first or reference NCCs. Fluorescence intensity data are obtained. The fluorescence intensity data are indicative of the presence of the first and reference NCCs.

Claims

1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC) having non-methylated cytosines, comprising:

contacting a first portion of the sample comprising the first NCC with a first compound to thereby provide a modified NCC, wherein non-methylated cytosines of the first NCC are replaced with a different base;

amplifying the modified NCC to obtain first PCR products;

preparing, from a second portion of the sample comprising the first NCC, a reference NCC having a sequence that differs from a sequence of the first PCR products at locations corresponding to the presence of the methylated cytosines in the first NCC;

subjecting the first PCR products to temperature gradient electrophoresis in the presence of the reference NCC;

irradiating the first PCR products and reference NCC with light to thereby generate a spectroscopic signal; and

converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC.

2. The method of claim 1, wherein the step of preparing a reference NCC comprises:

amplifying the second amount of the first NCC to prepare second PCR products; and

contacting the second PCR products with a first compound to thereby provide a second modified NCC, wherein non-methylated cytosines of the second PCR products are replaced with a different base.

3. The method of claim 1, wherein uracil is replaced by thymine during the step of amplifying the modified NCC.

4. The method of claim 1, wherein, prior to the irradiating step, the first PCR products and the reference NCC are combined to obtain at least one heteroduplex and at least one homoduplex.

5. The method of claim 1, wherein the amplifying step comprises contacting the first NCC with strand specific primers.

6. The method of claim 1, wherein the strand specific primers comprise a fluorescent tagging compound.

7. The method of claim 1, wherein the first compound comprises a bisulfite salt.

8. The method of claim 1, wherein the different base is uracil.

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

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

11. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC), comprising:

obtaining first PCR products formed by;

(a1) contacting the first NCC with a first compound to thereby provide a modified NCC in which non-methylated cytosines are replaced with a different base, and (a2) amplifying the modified NCC to obtain said first PCR products;

obtaining a reference NCC, the reference NCC being prepared from a second portion of the sample comprising the first NCC, the reference NCC having a sequence that differs from a sequence of the first PCR products at locations corresponding to the presence of the methylated cytosines in the first NCC;

subjecting the first PCR products and a reference NCC to temperature gradient electrophoresis,

irradiating the first PCR products and reference NCC with light to thereby generate a spectroscopic signal; and

converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC.

12. The method of claim 11, wherein preparing the the reference NCC comprises:

amplifying the second amount of the first NCC to prepare second PCR products; and

contacting the second PCR products with a first compound to thereby provide a second modified NCC, wherein non-methylated cytosines of the second PCR products are replaced with a different base.

13. The method of claim 11, wherein uracil is replaced by thymine during the step of amplifying the modified NCC.

14. The method of claim 11, wherein, prior to the irradiating step, the first PCR products and the reference NCC are combined to obtain at least one heteroduplex and at least one homoduplex.

15. The method of claim 11, wherein the amplifying step comprises contacting the first NCC with strand specific primers.

16. The method of claim 15, wherein the strand specific primers comprise a fluorescent tagging compound.

17. The method of claim 11, wherein the first compound comprises a bisulfite salt.

18. The method of claim 11, wherein the different base is uracil.

19. The method of claim 11, wherein the spectroscopic signals are fluorescence signals.

20. The method of claim 11, wherein the spectroscopic signals are absorbance signals.