Poly-primed amplification of nucleic acid sequences
AssigneeMolecular Staging, Inc.
InventorsStephen Kingsmore, R. Steven Wiltshire, Jeremy P. Lambert
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Abstract
Methods for the amplification of selected nucleic acids at high rate but with high specificity and control are disclosed using a secondary, tertiary, quaternary or higher order platform especially designed to amplify selected sequences within the primary product of linear or exponential rolling circle amplification and amplifying said sequences along with specialized detector or reporter molecules that serve to enhance the ability to detect the amplification products.
Claims43
A process for detecting nucleic acid sequences comprising:
- (a) contacting a single stranded non-circular oligonucleotide primer with an amplification target circle (ATC), said ATC comprising a segment complementary to at least one segment of said primer and wherein said at least one segment includes the 3′-terminus of said primer, under conditions promoting hybridization of said primer with said ATC to form a primer-ATC complex,
- (b) contacting said complex of (a) with a rolling circle amplification (RCA)-promoting enzyme under conditions promoting rolling circle amplification of said ATC from the 3′-terminus of said primer, wherein said conditions include the presence of a dNTP substrate bound to a reporter molecule, to form a tandem sequence DNA (TS-DNA) that incorporates said reporter molecule,
- (c) contacting said TS-DNA with a plurality of primers wherein said primers are bound at their 5′-terminus to a reactive molecule that binds to said reporter molecule and wherein said primers comprise a segment not complementary to said TS-DNA of (b),
- (d) contacting said plurality of primers with a plurality of ATCs each comprising a segment complementary to a segment of said plurality of primers of (c) wherein said segment of said plurality of primers includes the 3′-terminus thereof, under conditions promoting rolling circle amplification of said plurality of primers to form additional TS-DNA,
- (e) repeating steps (c) and (d) n times where n has a value of 0 or higher,
- (f) detecting the presence of said TS-DNA.
A process for amplifying a selected nucleic acid sequence comprising:
- (a) contacting a single stranded non-circular oligonucleotide primer with an amplification target circle (ATC), said ATC comprising a segment complementary to at least one segment of said primer and a segment not complementary to said primer of (a) and wherein said at least one segment of said primer includes the 3′-terminus of said primer, under conditions promoting hybridization of said primer with said ATC to form a primer-ATC complex,
- (b) contacting said complex of (a) with a rolling circle amplification (RCA)-promoting enzyme under conditions promoting rolling circle amplification of said ATC from the 3-terminus of said primer, wherein said conditions include the presence of one or more deoxynucleoside triphosphates (dNTPs), to form a tandem sequence DNA (TS-DNA),
- (c) contacting said TS-DNA with a plurality of primers wherein said primers comprise a segment complementary to at least a portion of said TS-DNA and a segment not complementary to said TS-DNA,
- (d) contacting said plurality of primers with a plurality of ATCs each comprising a segment complementary to a segment of said plurality of primers of (c) that includes the 3′-terminus thereof, under conditions promoting rolling circle amplification of said plurality of primers to form additional TS-DNA,
- (e) repeating steps (c) and (d) n times where n has a value of 0 or higher,
- thereby amplifying said selected nucleic acid sequence.
Dependencies are inferred from the claim text.