Fluorescence quenching azo dyes, their methods of preparation and use

A method for making an oligonucleotide, comprising introducing the compound of Formula 1 to an automated oligonucleotide synthesizer to chemically incorporate a quencher compound into the oligonucleotide:

• wherein R1-6 on the conjugated ring system are individually an electron withdrawing group, alkyl group, aryl group, hydrogen, heteroaryl group, or a five or six member ring structure formed from the R1, R2 pair, the R3, R4 pair, the R4, R5 pair, or the R5, R6 pair;
• and wherein R7 is an anilyl group of Formula 2:
• wherein L and L′ are the same or different and are a C1-10 group, —C1-10—O—R8, or —C1-10—O—R9, wherein C1-10 is a C1-10 alkyl group, and R8 and R9 are independently a hydrogen, trityl, alkoxytrityl, silyl, or phosphoramidite with the proviso that when L or L′ is ethyl, then the other is not hydroxyethyl, and
• wherein at least one of L or L′ is —C1-10—O—R8 or —C1-10—O—R9, and at least one of R8 and R9 is a phosphoramidite.