Terminating substrates for DNA polymerases

• US 20070248973A1
• Filed: 02/26/2007
• Published: 10/25/2007
• Est. Priority Date: 03/13/2006
• Status: Active Grant

• Current Assignee: Wallac Oy (Revvity, Inc.)
• Original Assignee: Wallac Oy (Revvity, Inc.)
• Inventors: Hovinen, Jari
• Granted Patent: US 7,482,444 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

This invention concerns novel terminating substrates for DNA polymerases. The substrates are nucleoside triphosphates labeled with lanthanide chelates.

Claims

1. A terminating substrate for DNA polymerases of formula (I)

Z-R-L-X (I)

wherein,

Z is a triphosphate anion or its organic or inorganic salt;

R is a recognizing moiety, which is a nucleoside comprising a base bound to a 2,3-dideoxyribose or its acyclic derivative; and

Z-R has the formula (II)

wherein the ribose ring optionally is replaced by an acyclic derivative in which one or both of the 2- and 3-carbon atoms from the ribose ring optionally are missing;

-L- is a linker; and

X is a lanthanide(III) chelate.

2. The substrate according to claim 1, wherein Z is an organic or inorganic salt of triphosphate, said salt is most preferably a sodium, lithium, calcium, potassium, magnesium, ammonium tributylammonium or a triethylammonium salt.

3. The substrate according to claim 1, wherein the recognizing moiety is a radical derived from the following seven structures;

4. The substrate according to claim 1, wherein the linker -L- is connected to C7 of 7-deazaadenine, C7 of 7-deazaguanine, C5 of cytosine, C5 of uracil, C3′

of 2′

-deoxyguanosine, C3′

2′

-deoxyadenosine, C3′

of 2′

-deoxycytidine or C3′

of thymidine.

5. The substrate according to claim 1, wherein the linker -L- is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkyl containing 1-12 carbon atoms, ethynediyl (—

C≡

C—

), ethylenediyl (—

C═

C—

);

ether (—

O—

), thioether (—

S—

), amide (—

CO—

NH— and

—

NH—

CO— and

—

CO—

NR′ and

—

NR′

—

CO—

), carbonyl (—

CO—

), ester (—

COO— and

—

OOC—

), disulfide (—

SS—

), diaza, (—

N═

N—

), thiourea (—

NH—

CS—

NH—

) or a tertiary amine (—

NR′

—

), where R′

represents an alkyl containing less than 5 carbon atoms.

6. The substrate according to claim 1, wherein the lanthanide chelate X is either luminescent or non-luminescent, and the lanthanide(III) ion is selected in such a way that each DNA nucleobase binding to the recognising moiety is detectable by one particular lanthanide(III) ion.

7. The substrate according to claim 6 where the lanthanide is selected from europium, terbium, samarium and dysprosium.

8. Use of the terminating substrate according to claim 1 in DNA sequencing for determining the base sequence wherein each base is identified by detecting the signal derived from the lanthanide chelate of said terminating substrate.