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Nested multigene MSP/DHPLC method for analyzing promoter hypermethylation status in clinical samples
 
Kevin K. Divine, Kieu C. Liechty, Kevin C. Crume, Steven A. Belinsky
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Supplementary Material
DivineSupp401 (.pdf)
Table 1. Nested Multiplex MSP Primers for p16, RASSF1A, and DAPK Genes


MSP, methylation-specific PCR.

DHPLC analyses were conducted using a DNASep® HT column (Transgenomic) under nondenaturing conditions at a temperature of 50°C with a 1.5 mL/min flow rate and UV detection. Under these conditions, PCR product separation is based on size not sequence. Resolution is limited to approximately 1% of the PCR product size. DHPLC analysis conditions for stages 1 and 2 nested three-gene MSP assays are listed in (Table 2). PCRs were loaded onto the WAVE autosampler, and 5-µL injections were used for each analysis. DHPLC analysis of both stage 1 and 2 products required <6 min/sample. Typical DHPLC detection of stages 1 and 2 unmethylated MSP products are shown in (Figure 1). It should be noted that given the heterogeneity of biological samples, verification of target gene amplification is normally accomplished by the presence of a stage 1 PCR product. Stage 2 unmethylated primers are typically used only when analyzing extensively degraded DNA as found in some formalin-fixed paraffin-embedded tissue samples.

Figure 1.


Denaturing high-performance liquid chromatography (DHPLC) chromatogram of stage 1 and stage 2 unmethylated methylation-specific PCR (MSP) products. Elution order proceeds from smallest to largest MSP product. (A) Stage 1 elution times are: DAPK, 2.33 min; RASSF1A, 2.73 min; and p16, 3.04 min. (B) Sputum (SP) sample #8 (SP#8) stage 2 unmethylated DHPLC chromatogram. Elution times are: DAPK, 2.07 min; p16, 2.64 min; and RASSF1A, 2.83 min. (C) Table listing the retention times and corresponding peak heights in millivolts of the DHPLC analysis of SP#8 stage 2 unmethylated nested three-gene stage 2 MSP products.
Table 2. DHPLC Analysis Gradients


DHPLC, denaturing high-performanc liquid chromato raphy. Time is in minu es. A, % uffer A 0.1 M trieth l mmon M triethylammonium acetate); %B, % buffer B (0.1 M triethylammonium acetate, 25% acetonitrile).

One-hundred percent concordance was observed in the detection of methylated stage 2 MSP products between the nested single-gene MSP/agarose gel electrophoresis method and the nested three-gene MSP/DHPLC method. An example of MSP product separation using each method is shown in (Figure 2), A–F. DHPLC-based separation has better resolution and increased sensitivity for detecting methylated PCR products compared with agarose gel electrophoresis ((Figure 2)). For example, sputum samples 5 and 8 are methylated for both p16 and DAPK. Resolving the 8-bp size difference between stage 2 PCR products is difficult using agarose gel electrophoresis, while this difference is clearly discerned by DHPLC. The increased resolution of DHPLC makes it easier to separate and identify methylated MSP products. This is essential in accurately determining the promoter methylation status of individual genes in a multiplex reaction. Additionally, as demonstrated in (Figure 2), E and F, DHPLC analysis of MSP products obtained using a formalin-fixed paraffin-embedded DNA template do not present added difficulties. Compared with conventional nested single-gene MSP, a nested multigene MSP/DHPLC method requires less time, reagents, and sample DNA to analyze the gene promoter methylation status of clinical samples from a variety of tissues. The ability to identify methylated MSP products using UV detection also eliminates the cost associated with fluorescence probes required for other nongel-based MSP methods such as TaqMan®. Savings realized are proportional to the number of genes present in the nested multigene MSP and/or the number of gene products analyzed per DHPLC chromatogram. To further increase throughput and savings, we are modifying the multigene MSP/DHPLC method to simultaneously detect promoter methylation of four genes (Pax5α, Pax5β, GATA4, and GATA5) ((Figure 3)). The Pax5 and GATA gene families encode transcription factors implicated in carcinogenesis (10,11). All four genes are silenced by aberrant promoter methylation in lung cancer (10,12). The conditions for the nested four-gene MSP/DHPLC method are available in the supplementary material available online at www.BioTechniques.com .

Figure 2.


Detection of stage 2 methylated products. (A) Agarose gel electrophoresis analysis of methylated nested three-gene stage 2 methylation-specific PCR (MSP) products. MSP product sizes are listed on the right of the gel, and corresponding gene identities are listed on the left side of the gel. WB represents the water blank. H2009, MCF7, and Calu6 are cell line controls for the different stage 2 MSP gene products. Sputum samples (SP) #1–#8 are from smokers at risk for lung cancer. Breast tumor (BT) samples BT#1 and BT#2 are from formalin-fixed paraffin-embedded breast tumors. (B) SP#2 denaturing high-performance liquid chromatography (DHPLC) chromatogram illustrating resolution of methylated p16 and RASSF1A MSP products. (C) SP#5 DHPLC chromatogram demonstrating resolution of p16 and DAPK MSP products of similar size and similar MSP product quantity. (D) SP#8 DHPLC chromatogram demonstrating resolution of p16 and DAPK MSP products of similar size with large differences in MSP product quantity. (E) BT #1 DHPLC chromatogram illustrating resolution of p16 and RASSF1A MSP products obtained from formalin-fixed paraffin-embedded tissues. (F) BT #2 DHPLC chromatogram illustrating resolution of DAPK and RASSF1A MSP products obtained from formalin-fixed paraffin-embedded tissues. (G) Compilation of DHPLC results from an analysis of methylated nested three-gene stage 2 MSP. The same SP samples were analyzed using both agarose gel electrophoresis and DHPLC. The Table lists retention times and corresponding peak heights in millivolts.

Figure 3.


Detection of nested three-gene and four-gene stage 2 methylation-specific PCR (MSP) methylated products. (A) Composite denaturing high-performance liquid chromatography (DHPLC) chromatogram containing three methylated stage 2 MSP products (p16, DAPK, and RASSF1A) obtained from the nested multigene MSP method. The sample was produced via mixing methylated cell line stage 2 MSP products. (B) DHPLC chromatogram containing four methylated stage 2 MSP products (Pax5β, GATA4, Pax5α, GATA5) obtained from the nested multigene MSP method using DNA from the MCF7 cell line. Elution times are: Pax5β, 2.00 min; GATA4, 2.26 min; Pax5α, 2.51 min; and GATA5, 2.87 min.

We have described a nested multigene MSP/DHPLC method to simultaneously examine gene promoter hypermethylation of three genes. Although this method was developed to detect multiple gene methylation events in complex biological samples, quantitation of methylation levels can be accomplished by determining the ratio of methylated to unmethylated DNA as reflected by individual peak heights expressed in millivolts ((Figure 1)B and (2)C). This method will aid in the rapid and economical assembly and validation of gene panels involved in cancer initiation, progression, and malignant transformation. Once validated, biomarkers based on these gene panels can be used in the early detection, diagnosis, and prognosis of multiple types of cancer.

Acknowledgments

This work was supported by a National Institutes of Health (NIH) grant from the National Cancer Institute Specialized Programs of Research Excellence (NCI SPORE) P50 CA58184, CA97356, and a research grant from OncoMethylome Sciences, Inc.)

Competing Interests Statement

Dr. Belinsky is a consultant to OncoMethylome Sciences. Under a licensing agreement between Lovelace Respiratory Research Institute (LRRI) and OncoMethylome Sciences, nested MSP was licensed to OncoMethylome Sciences, and Dr. Belinsky is entitled to a share of the royalties received by LRRI from sales of the licensed technology. LRRI, in accordance with its conflict-of-policies, is managing the terms of these arrangements. The remaining authors declare no competing interests.

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