US 12,448,650 B2
Quantitative multiplex methylation specific PCR method-CMethDNA, reagents, and its use
Saraswati Sukumar, Columbia, MD (US); Mary Jo Fackler, Hunt Valley, MD (US); Wei Wen Teo, Johor Bahru Johor (MY); Zoila Areli Lopez Bujanda, Bethesda, MD (US); and Antonio Wolff, Baltimore, MD (US)
Assigned to The Johns Hopkins University, Baltimore, MD (US)
Filed by The Johns Hopkins University, Baltimore, MD (US)
Filed on Oct. 14, 2019, as Appl. No. 16/601,269.
Application 16/601,269 is a division of application No. 14/402,434, granted, now 10,450,609, issued on Oct. 22, 2019, previously published as PCT/US2013/042198, filed on May 22, 2013.
Claims priority of provisional application 61/650,083, filed on May 22, 2012.
Prior Publication US 2020/0190586 A1, Jun. 18, 2020
Int. Cl. C12Q 1/6883 (2018.01); C12P 19/34 (2006.01); C12Q 1/686 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6883 (2013.01) [C12P 19/34 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6886 (2013.01); C12Q 2523/125 (2013.01); C12Q 2525/161 (2013.01); C12Q 2525/185 (2013.01); C12Q 2525/204 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/154 (2013.01); C12Q 2600/16 (2013.01)] 8 Claims
 
1. A synthetic polynucleotide standard (STDgene) for use in real-time PCR (RT-PCR) comprising an oligonucleotide having 5′ and 3′ end nucleotide sequences which are homologous to the 5′ and 3′ end nucleotide sequences of an endogenous methylated TARGETgene of interest, wherein the intervening oligonucleotide sequence between the 5′ and 3′ end nucleotide sequences in the STDgene comprises a lambda phage DNA sequence, and the number of intervening bases between the 5′ and 3′ end nucleotide sequences in the STDgene oligonucleotide is equivalent to the entire length of a region of interest, defined by a particular 5′ and 3′ sequence, within the TARGETgene of interest, wherein the STDgene oligonucleotide is selected from: SEQ ID NOs: 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, or 240.