Method for accurate assessment of DNA quality after bisulfite treatment
The present invention is directed to methods useful for determining DNA quality after bisulfite treatment. The methods include a PCR-based assay, which allows ab-initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.
1. A method to determine the maximum amplicon size for a nucleic acid target region in nucleic acid of a sample after bisulfite treatment that will yield accurate quantitative measurements, comprising:
a) adding to the sample a control nucleic acid having a known methylation ratio, thereby generating a sample comprising i) sample nucleic acid and ii) control nucleic acid;
b) treating the sample with bisulfite;
c) performing PCR using a primer set that generates at least two amplicons from a control region in the nucleic acid target region, wherein the amplicons vary in length and share a subset of CpG sites;
d) determining the methylation status for at least three CpG sites that are common to all of the amplicons of step (c);
e) based on methylation status determined in step (d) determining methylation ratios for the at least three CpG sites that are common to all of the amplicons with respect to the methylation ratio of the control nucleic acid;
f) determining amplification success and statistical variability of methylation ratios of step (e) for the at least three CpG sites that are common to all of the amplicons; and
g) selecting amplicon sizes that yield detectable amplification products and low statistical variability of methylation ratio for a given sample.
2. The method of claim 1 , further comprising modifying at least one of the methylation conditions to introduce variable methylation conditions and selecting methylation conditions that are optimal across a range of amplicon sizes for nucleic acid in a sample.
3. The method of claim 2 , wherein the methylation conditions are selected from the group consisting of sample handling methods, bisulfite treatment methods, PCR conditions, methylation-related biochemistry methods and detection methods.
4. The method of claim 3 , wherein the PCR conditions are selected from the group consisting of temperature, incubation time and PCR primer concentration.
5. The method of claim 1 , wherein the bisulfite concentration of step b) is the same or substantially the same as the bisulfite concentration of a target assay.
6. The method of claim 1 , wherein the PCR conditions of step c) are the same or substantially the same as the PCR conditions of a target assay.
7. The method of claim 1 , wherein the primers of step c) hybridize to binding sites that are free of CpG sites.
8. The method of claim 1 , wherein the PCR reaction of step c) is done in a single reaction.
9. The method of claim 1 , wherein the PCR reaction of step c) amplifies at least 3 amplicons from a control region in the nucleic acid target region.
10. The method of claim 1 , wherein the PCR reaction of step c) amplifies at least 4 amplicons from a control region in the nucleic acid target region.
11. The method of claim 1 , wherein the shortest amplicon is at least 100 base pairs.
12. The method of claim 1 , wherein the longest amplicon is no more than 900 base pairs.
13. The method of claim 1 , wherein the amplicons are increased in increments between about 100 and 150 base pairs.
14. The method of claim 1 , wherein the amplicons cover substantially the same region.
15. The method of claim 1 , wherein the control region in the sample nucleic acid comprises at least 3 CpG sites, wherein each CpG site has a known methylation ratio.
16. The method of claim 1 , wherein the control region is the promoter region of IGF2/H19.
17. The method of claim 1 , wherein the control nucleic acid comprises nucleic acid from a distinguishable nucleic acid source.
18. The method of claim 17 , wherein the control nucleic acid comprises non-human nucleic acid.
19. A method to determine methylation conditions which yield results more accurate across a range of amplicon sizes for a nucleic acid target region in nucleic acid of a sample, comprising:
a) adding to the sample a control nucleic acid having a known methylation ratio, thereby generating a sample comprising i) sample nucleic acid and ii) control nucleic acid;
b) treating the sample with bisulfite;
c) performing PCR using a primer set that generates at least two amplicons from a control region in the nucleic acid target region, wherein the amplicons vary in length and share a subset of CpG sites;
d) determining the methylation status for at least three CpG sites that are common to all of the amplicons of step (c);
e) based on methylation status determined in step (d) determining methylation ratios for the at least three CpG sites that are common to all of the amplicons with respect to the methylation ratio of the control nucleic acid;
f) determining amplification success and statistical variability of methylation ratios of step (e) for the at least three CpG sites that are common to all of the amplicons;
g) modifying at least one of the methylation conditions to introduce variable methylation conditions, wherein the methylation conditions are selected from the group consisting of sample handling, bisulfite treatment methods, amplification conditions, and methylation detection methods;
h) repeating steps (a) to (f) to determine amplification success and statistical variability of methylation ratios for the at least three CpG sites that are common to all of the amplicons using the variable methylation conditions of step (g);
i) comparing the amplification success and statistical variability of methylation ratios of step (f) and step (h) for the at least three CpG sites that are common to all of the amplicons; and
j) selecting methylation conditions that are optimal across a range of amplicon sizes and yield detectable amplification products and low statistical variability of methylation ratio for a given sample.