GLAD-PCR-Assay - new method for DNA methylation analisys

GLAD-PCR assay (GlaI hydrolysis and Ligation Adapter Dependent PCR) is a new and unique method for study of DNA methylation.

The method allows to determine 5'-R(5mC)GY-3' site in selected position of human and mammalian genomic DNA.

Today an abnormal methylation of regulation regions (promoter and/or first exon) of genes was shown at initial stage of several deceases such as cancer, cardiovascular decease, diabetes and some others.

This abnormal de novo DNA methylation is performed by DNMT3A and DNMT3B DNA methyltransferases. These enzymes recognize and methylate site 5’-RCGY-3’ with formation of 5’-R(5mC)GY-3’/3’YG(5mC)R-5’[1]. Recently discovered site-specific methyl-directed DNA-endonuclease GlaI recognizes exactly this DNA sequence 5’-R(5mC)^GY-3’/3’YG^(5mC)R-5’ and cleaves it as indicated by symbols ^ [2].

GLAD PCR assay is based on GlaI application, includes three steps and is carried out in one tube [3] (see animation below):

  1. GlaI hydrolysis of studied DNA
  2. the universal adapter ligation
  3. subsequent real-time PCR with Taqman probe.
  4. NO BISULFITE CONVERSION!

A new method of GLAD PCR assay is used to determine a selected 5'-R(5mC)GY-3' site in a human/mammalian DNA at minimal quantities (5 and more copies) in a presence of excess of unmethylated DNA.

GLAD PCR analysis has been used to study an aberrant methylation of regulatory regions of RARB, CEBPD, ESR1, ELMO1 [4, 6, 10] and other genes in malignant tissues and cell lines.

GLAD PCR analysis is developed for epigenetic DNA diagnostics in routine laboratory and clinical practice.

de novo DNA methylation

GLAD-PCR assay stages

Innovative aspects and main advantages

In comparison with other methylation detection methods GLAD-PCR has strong advantages:
  • Simple – 3 easy steps in one tube
  • Requires only real time PCR-machine
  • Quick - only 3-4 hours
  • Sensitive – detects from 6 copies of methylated DNA
All these factors make GLAD-PCR-Assay very attractive to apply in epigenetic diagnostics

GLAD-PCR assay kit

A new kit is designed for 200 GLAD-PCR reactions in two 96-wells PCR plate. All reagents are included except primers and TaqMan probe. The sequences of TaqMan Probe and primers are determined by a customer based on DNA sequence around the R(5mC)GY site of interest.

Reaction requires:

  • sample DNA
  • genomic primer and TaqMan probe designed for R(5mC)GY site of interest
  • hybrid primer (includes constant part complimentary to the universal adapter and tetranucleotide part complimentary to DNA at the point of GlaI hydrolysis).

GLAD-PCR assay kit is available for orders on our site.

Please download GLAD-PCR assay kit instruction manual



Primers and TaqMan probes

For GLAD-PCR assay reaction you will need 1 TaqMan probe and 2 primers: genomic and hybrid.

Primers and TaqMan probes Genomic primer and TaqMan probe located near 5’-R(5mC)^GY-3’ site of interest are designed as usual [4].

Hybrid primer is a DNA sequence 5’-CCTGCTCTTTCATCGGYNN-3’, wherein 5’-end of
15-nucleotide primer corresponds to universal adapter and tetranucleotide part at the 3’-end (underlined) is complementary to the genomic sequence at GlaI hydrolysis point. This structure implies the existence of 32 variants of hybrid primers corresponding to different possible terminal sequences after GlaI hydrolysis of all possible variants of NNR(5mC)^GY sequence.

All primers and probes may be ordered at SibEnzyme along with the GLAD-PCR assay kit order. Otherwise, you can order a synthesis of primers and TaqMan probe somewhere else.

GLAD PCR Assay of two R(5mC)GY sites in human genome are used as an example of the method application.
Mixes of TaqMan Probes and primers for analysis of only these two R(5mC)GY sites are included:

  1. "Control URB1 mix (primers + TaqMan probe)" (p.16) is provided for analysis of A(5mC)GT site in regulation region of URB1 gene. Position of this site (according to a human  genome assembly GRCh38/hg38) is 32334291-32334294 in chromosome 21.  
  2. "Control CEBPD mix (primers + TaqMan probe)" (p.17) is provided for analysis of G(5mC)GC site in regulation region of CEBPD gene. Position of this site (according to a human  genome assembly  GRCh38/hg38) is 47738502-47738505 in chromosome 8.

Method animation and control experiment demonstration


GLAD-PCR assay presentation at scientific conferences:

  1. 43th ISOBM annual congress, September, 2016, Chicago, USA, oral presentation GLAD-PCR assay of DNA methylation markers associated with colorectal cancer
  2. Global Cancer Summit 2015, Bangalore, India, oral presentation "GLAD-PCR assay of R(5mC)GY sites in aberrantly methylated regulation regions of tumor-suppression genes in colorectal cancer"
  3. Cancer Epigenomics 2015, San-Francisco, USA, poster presentation "Determination of R(5mC)GY sites in regulation regions of tumor-suppression genes by GLAD-PCR assay in colorectal cancer"
  4. Cell Symposia Cancer Epigenomics, Barselona, Spain, October, 2013, Poster presentation GLAD PCR analysis of aberrant DNA methylation in cancer

Related PDF files

  1. Methyl-directed DNA Endonucleases
  2. GLAD-PCR assay
  3. GLAD PCR assay Kit
  4. Early Cancer Detection
  5. Determination of R(5mC)GY sites in regulation regions of tumor suppression genes by GLAD-PCR assay in colorectal cancer

Related articles and patents:

  1. Handa, V., and Jeltsch A. “Profound sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome” // 2005, J. Mol. Biol. 348, 1103-1112
  2. Tarasova G.V., Nayakshina T.N., Degtyarev S.Kh. Substrate specificity of new methyl-directed DNA endonuclease GlaI // BMC Molecular Biology 2008, 9:7
  3. Kuznetsov V.V., Abdurashitov M.A., Akishev A.G., Degtyarev S.Kh. Method of determining nucleotide sequence Pu(5mC)GPy at predetermined position of continuous DNA // Patent RU 2525710 C1
  4. A.G. Akishev, M.A. Abdurashitov, V.L. Sitko, N.A. Netesova, I.F. Radaeva, E.A. Nechaeva, S.Kh. Degtyarev “GLAD-PCR assay of selected R(5mC)GY sites in URB1 and CEPBD genes in human genome” // Res J Pharm Biol Chem Sci, 8(1): pp.465-475, 2017
  5. A.A. Evdokimov , N.A. Netesova , N.A. Smetannikova , M.A. Abdurashitov, A.G. Akishev , B.S. Malyshev , E.S. Davidovich , V.V. Fedotov , V.V. Kuznetsov , Y.D. Ermolaev , A.B. Karpov , A.E. Sazonov , R.M. Tahauov, S.Kh. Degtyarev GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer // Biol Med (Aligarh) , 8:7(2016)
  6. A.A. Evdokimov, N.A. Netesova, N.A. Smetannikova, M.A. Abdurashitov, A.G. Akishev, E.S. Davidovich, Yu.D. Ermolaev, A.B. Karpov, A.E. Sazonov, R.M. Tahauov, S.Kh. Degtyarev Application of GLAD-PCR assay for determination of the methylation sites in the regulatory regions of tumor-supressors gene ELMO1 and ESR1 in colorectal cancer // Problems in oncology, #1, 2016 p.116-120
  7. Alexander G. Akishev, Danila A. Gonchar, Murat A. Abdurashitov and Sergey Kh. Degtyarev Epigenetic typing of human cancer cell lines by BlsI- and GlaI-PCR assays // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 3, pp 5-16, 2011
  8. D. A. Gonchar, A. G. Akishev, S. Kh. Degtyarev BlsI- and GlaI-PCR assays – a new method of DNA methylation study // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.6, No 1, pp 5-12, 2010
  9. V.A. Chernukhin, T.N. Najakshina, M.A. Abdurashitov, J.E. Tomilova, N.V. Mezentzeva, V.S. Dedkov, N.A. Mikhnenkova, D.A. Gonchar, S.Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5’-G(5mC)^GC-3’ // Biotechnologia (russ.). 2006. N 4. P. 31-35
  10. A.A. Evdokimov, M.V. Kulak, N.A. Netesova, N.A. Smetannikova, M.A., Abdurashitov, A.G. Akishev, B.S. Malyshev, E.S. Davidovich, V.V. Fedotov, A.B. Karpov, S.Kh. Degtyarev GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer // Tumor Biology, Vol.37, supplement 1, Sept. 2016, s6-s7

 

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