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’. 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 ^ .
GLAD PCR assay is based on GlaI application, includes three steps and is carried out in one tube  (see animation below):
GlaI hydrolysis of studied DNA
the universal adapter ligation
subsequent real-time PCR with Taqman probe.
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.
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.
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).
For GLAD-PCR assay reaction you will need 1 TaqMan probe and 2 primers: genomic and hybrid.
Genomic primer and TaqMan probe located near 5’-R(5mC)^GY-3’ site of interest are designed as usual .
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:
"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.
"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: