Methods for evaluating the methylation status of a polynucleotide

• US 20100105063A1
• Filed: 12/29/2009
• Published: 04/29/2010
• Est. Priority Date: 07/02/2007
• Status: Active Grant

• Current Assignee: Euclid Diagnostics LLC
• Original Assignee: Euclid Diagnostics LLC
• Inventors: Brikun, Igor, Freije, Wadiha
• Granted Patent: US 8,361,724 B2
• 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 2521/125 Methyl transferase, i.e. methylase
• 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

The invention provides methods related to evaluating the methylation status of a polynucleotide that includes an internal control.

Claims

1. A method for analyzing the methylation status of a target sequence in a sample with an internal control for detecting the presence of the target sequence, the method comprising:

(a) providing a sample that includes DNA;

(b) optionally, purifying the DNA from the sample to thereby produce purified DNA;

(c) optionally, amplifying the DNA and simultaneously copying the methylation pattern of the DNA to thereby produce amplified DNA;

(d) treating the sample, purified DNA, or amplified DNA with a methylase that preferentially methylates at one or more control sequence nucleotides in a target sequence; and

(e) assaying the sample, purified DNA, or amplified DNA for the methylation status of (i) the one or more control sequence nucleotides and (ii) one or more CpG dinucleotides in the target sequence;

wherein (a′

) methylation at the one or more control sequence nucleotides indicates the presence of the target sequence in the sample, and the presence or absence of methylation at the one or more CpG dinucleotides indicates the presence or absence, respectively, of methylation at the corresponding dinucleotides in the sample, or (b′

) absence of methylation at the one or more control sequence nucleotides indicates the absence of the target sequence in the sample, purified DNA, or amplified DNA and/or a technical failure.

2. The method of claim 1, wherein (e) comprises using terminator-coupled linear amplification suitable for generating extension products that indicate (i) the methylation status of the one or more control sequence nucleotides in the treated sample, purified DNA, or amplified DNA and (ii) the methylation status of one or more CpG dinucleotides in the target sequence;

and wherein (a′

) methylation at the one or more control sequence nucleotides indicates the presence of the target sequence in the sample, and the presence or absence of methylation at the one or more CpG dinucleotides indicates the presence or absence, respectively, of methylation at the corresponding dinucleotides in the sample, or (b′

) absence of methylation at the one or more control sequence nucleotides indicates the absence of the target sequence in the sample, purified DNA, or amplified DNA and/or failure of the terminator-coupled linear amplification reaction.

3. The method of claim 1, wherein the sample is a bodily fluid selected from the group consisting of blood, blood plasma, blood serum, urine, sputum, ejaculate, semen, tears, sweat, saliva, lymph fluid, bronchial lavage, pleural effusion, peritoneal fluid, meningal fluid, amniotic fluid, glandular fluid, fine needle aspirates, nipple aspirate fluid, spinal fluid, conjunctival fluid, vaginal fluid, duodenal juice, pancreatic juice, pancreatic ductal epithelium, pancreatic tissue bile, and cerebrospinal fluid.

4. The method of claim 1, wherein the sample includes less than 10 nanograms of DNA.

5. The method of claim 4, wherein the sample includes fewer than 150 contiguous nucleotides that include the target sequence.

6. The method of claim 4, wherein the sample is urine and the target sequence includes at least a portion of a CpG island having a methylation status that is associated with prostate cancer.

7. The method of claim 6, wherein the CpG island is associated with a gene selected from the group consisting of GSTP1, GPX3, GSTM1, GSTM4, CSMD1, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, SFRP1, SFRP2, DKK3, PTGS2, CDKN1C/p57, RASSF1, GPR62, NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1, MMP9, TNFSF11, RHOD, and LRRC49.

8. A method for evaluating the density of DNA methylation in a target sequence with an internal control for detecting the presence or absence of the target sequence, the method comprising:

(a) providing a sample that includes DNA;

(b) optionally, purifying the DNA to thereby produce purified DNA;

(c) optionally, amplifying the DNA and simultaneously copying the methylation pattern of the DNA to thereby produce amplified DNA;

(d) treating the sample, purified DNA, or amplified DNA with a methylase that preferentially methylates at one or more control sequence nucleotides in a target sequence;

(e) using terminator-coupled linear amplification to generate extension products from the treated sample, purified DNA, or amplified DNA;

(f) assaying for the lengths of the extension products of (e) that indicate the methylation status of (i) the one or more control sequence nucleotides and (ii) more than one CpG dinucleotide in the target sequence to thereby determine a number of methylated CpG dinucleotides in the target sequence;

wherein (a′

) methylation at the one or more control sequence nucleotides indicates the presence of the target sequence in the sample, and the number of methylated CpG dinucleotides indicates the density of DNA methylation in the target sequence, or (b′

) absence of methylation at the one or more control sequence nucleotides indicates the absence of the target sequence in the sample, purified DNA, or amplified DNA and/or failure of the terminator-coupled linear amplification reaction.

9. A method of evaluating the fraction of polynucleotides comprising a methylated target sequence with an internal control for detecting the presence of the target sequence, the method comprising:

(a) providing a sample that includes DNA;

(b) optionally, purifying the DNA to thereby produce purified DNA;

(c) optionally, amplifying the DNA and simultaneously copying the methylation pattern of the DNA to thereby produce amplified DNA;

(d) treating the sample, purified DNA, or amplified DNA with a methylase that preferentially methylates at one or more control sequence nucleotides in a target sequence;

(e) using terminator-coupled linear amplification suitable for generating extension products that indicate the methylation status of (i) the one or more control sequence nucleotides and (ii) one or more CpG dinucleotides in the target sequence in the treated sample, purified DNA, or amplified DNA.

(f) assaying for the length and amount of each of the one or more extension products of (e) that indicate the methylation status of one or more CpG dinucleotides in the target sequence; and

(g) comparing the amount of each assayed extension product of (f) to a corresponding amount in a standard generated from known amounts of methylated and unmethylated template;

wherein (a′

) methylation at the one or more control sequence nucleotides indicates the presence of the target sequence in the original sample and each corresponding amount in the standard indicates the fraction of polynucleotides in the sample, purified DNA, or amplified DNA that comprises the corresponding methylated CpG dinucleotide in the target sequence, or (b′

) absence of methylation at the one or more control sequence nucleotides indicates the absence of the target sequence in the sample, purified DNA, or amplified DNA and/or failure of the terminator-coupled linear amplification reaction.

10. A method of preparing a sample for methylation analysis, which comprises

(a) providing a sample with DNA;

(b) optionally, purifying the DNA to thereby produce purified DNA;

(c) optionally, amplifying the DNA and copying the methylation pattern of the DNA to thereby produce amplified DNA;

(d) treating the sample, purified DNA, or amplified DNA with a methylase that preferentially methylates cytosine residues within a recognition sequence larger than 2 nucleotides;

wherein the sample is a bodily fluid selected from the group consisting of blood, blood plasma, blood serum, urine, sputum, ejaculate, semen, tears, sweat, saliva, lymph fluid, bronchial lavage, pleural effusion, peritoneal fluid, meningal fluid, amniotic fluid, glandular fluid, fine needle aspirates, nipple aspirate fluid, spinal fluid, conjunctival fluid, vaginal fluid, duodenal juice, pancreatic juice, pancreatic ductal epithelium, pancreatic tissue bile, and cerebrospinal fluid.

11. The method of claim 10, wherein the preferentially methylated cytosine is not within a CpG dinucleotide.

12. The method of claim 10, wherein the sample is plasma.

13. The method of claim 10, wherein the sample is urine and the method includes (c) amplifying the DNA to produce amplified DNA and (d) treating the amplified DNA with a methylase that preferentially methylates cytosine residues within a recognition sequence larger than 2 nucleotides.

14. The method of claim 13, wherein the sample, purified DNA, or amplified DNA is screened for the methylation status of one or more CpG island.

15. The method of claim 14, wherein the sample is urine and the methylation status of the CpG island is associated with prostate cancer.

16. The method of claim 15, wherein the CpG island is associated with a gene selected from the group consisting of GSTP1, GPX3, GSTM1, GSTM4, CSMD1, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, SFRP1, SFRP2, DKK3, PTGS2, CDKN1C/p57, RASSF1, GPR62, NRG1, ADRB3, GFRA2, KIF13B, RET, GPR147, NEUROG3, PALD, HEMK1, FGF4, HTR1A, RNF180, DKFZP5640822, ZNF596, LOC441320, TDH, FLJ36980, FGF20, EFHA2, ASAH1, NODAL, LOC399783, ISL2, KIFC2, C20orf23, GFRA1, GPX7, DKK2, NTN1, MMP9, TNFSF11, RHOD, and LRRC49.

17. The method of any one of claims 10, wherein the methylase recognition sequence comprises a sequence selected from the group consisting of AGCT, GGCC, GCNGC, GCGC, CCGG, GCGC, CCGG, and CCGG.