Detection of short homopolymeric repeats

Abstract

The present application relates to detection of changes in the number of nucleotides in short homopolymeric nucleic acid repeats, in particular in short homopolymeric microsatellites, for example for the purpose of diagnosing microsatellite instability (MSI) and/or mismatch repair (MMR-) deficiency in tumors. Accordingly, methods are provided for detecting changes in the number of nucleotides present in short homopolymeric nucleotide repeat sequences as well as kits and cartridges for automated detection of said changes.

1 - 15 . (canceled) 
     
     
         16 . A cartridge engageable with an automated system for determining a melting curve for a nucleic acid duplex formed by a molecular beacon oligonucleotide probe hybridized to a target nucleic acid sequence, the melting curve determined as a function of a change in fluorescence of the molecular beacon oligonucleotide probe versus temperature, the cartridge comprising:
 (a) a sample compartment for receiving a biological sample comprising the target nucleic acid sequence;   (b) the molecular beacon oligonucleotide probe, wherein the molecular beacon oligonucleotide probe comprises:
 (i) a loop region that hybridizes to the target nucleic acid sequence, the loop region comprising 18-30 nucleotides, the 18-30 nucleotides comprising a homopolymeric nucleotide sequence of 10-15 repeated nucleotides, the homopolymeric nucleotide sequence flanked at its 5′ end and 3′ end by non-homopolymeric sequences comprising a nucleotide different than the nucleotide of the homopolymeric nucleotide sequence; 
 (ii) a stem region formed by 5-7 nucleotides flanking the 5′ end and 3′ end of the loop region, wherein the 5-7 nucleotides are complementary to each other and form a stem when the molecular beacon oligonucleotide probe is not hybridized to the target nucleic acid sequence; and 
 (iii) a fluorophore and a quencher attached at opposite ends of the molecular beacon oligonucleotide probe; and 
   (c) a polymerase having 3′-5′ exonuclease activity.   
     
     
         17 . The cartridge of  claim 16 , wherein each non-homopolymeric flanking sequence is 5-7 nucleotides in length. 
     
     
         18 . The cartridge of  claim 16 , wherein each non-homopolymeric flanking sequence is 5-6 nucleotides in length. 
     
     
         19 . The cartridge of  claim 16 , wherein the homopolymeric nucleotide sequence of 10-15 repeated nucleotides consist of: homopolymeric A; homopolymeric G;
 homopolymeric T; or homopolymeric C.   
     
     
         20 . The cartridge according to  claim 16 , wherein the cartridge comprises at least two molecular beacon probes, wherein each of the at least two molecular beacon probes comprises a different homopolymeric nucleotide sequence. 
     
     
         21 . The cartridge according to  claim 20 , wherein the different homopolymeric nucleotide sequences differ in length and/or homopolymeric sequence. 
     
     
         22 . The cartridge according to  claim 16 , comprising a plurality of molecular beacon probes, wherein each of the plurality of molecular beacon probes comprises non-homopolymeric flanking sequences that are complementary to the target nucleic acid; and wherein each of the plurality of molecular beacon probes comprises different non-homopolymeric flanking sequences. 
     
     
         23 . The cartridge according to  claim 16 , further comprising primers for amplifying the nucleic acid sequence comprising the target homopolymeric repeat sequence. 
     
     
         24 . The cartridge of  claim 16 , further comprising a thermocycling quantitative polymerase chain reaction (qPCR) compartment comprising at least one transparent wall. 
     
     
         25 . The cartridge of  claim 24 , wherein the thermocycling qPCR compartment is fluidically connected to the sample compartment and downstream of the sample compartment. 
     
     
         26 . The cartridge of  claim 25 , wherein the thermocycling qPCR compartment comprises the molecular beacon oligonucleotide probe, the polymerase, and at least one PCR primer pair. 
     
     
         27 . A system comprising:
 (a) a cartridge comprising:
 (1) a sample compartment for receiving a biological sample comprising a target nucleic acid sequence; 
 (2) a thermocycling quantitative polymerase chain reaction (qPCR) compartment comprising at least one transparent wall;
 wherein the thermocycling qPCR compartment is fluidically connected to the sample compartment and downstream of the sample compartment; 
 wherein the thermocycling qPCR compartment comprises: 
 (i) at least one PCR primer pair; 
 (ii) a polymerase having 3′-5′ exonuclease activity; 
 (iii) at least one molecular beacon oligonucleotide probe, wherein the molecular beacon oligonucleotide probe comprises:
 a. a loop region that hybridizes to the homopolymeric target nucleic acid sequence, the loop region comprising 18-30 nucleotides, the 18-30 nucleotides comprising a homopolymeric nucleotide sequence of 10-15 repeated nucleotides, the homopolymeric nucleotide sequence flanked at its 5′ end and 3′ end by non-homopolymeric sequences comprising a nucleotide different than the nucleotide of the homopolymeric nucleotide sequence; 
 b. a stem region formed by 5-7 nucleotides flanking the 5′ end and 3′ end of the loop region, wherein the 5-7 nucleotides are complementary to each other and form a stem when the molecular beacon oligonucleotide probe is not hybridized to the target nucleic acid sequence; and 
 c. a fluorophore and a quencher attached at opposite ends of the molecular beacon oligonucleotide probe; 
 
 
 (b) a device comprising (i) a cartridge receiver,(ii) a thermocycler, (iii) fluorescence reader, and (iv) software programmed to calculate a melting curve for a nucleic acid duplex formed by the molecular beacon oligonucleotide probe hybridized to the target nucleic acid sequence, the melting curve determined as a function of a change in fluorescence of the molecular beacon oligonucleotide probe versus temperature; 
 wherein the cartridge is engaegable with the device at the cartridge receiver. 
   
     
     
         28 . The system of  claim 27 , wherein each non-homopolymeric flanking sequence of the molecular beacon probe is 5-7 nucleotides in length. 
     
     
         29 . The system of  claim 27 , wherein each non-homopolymeric flanking sequence of the molecular beacon probe is 5-6 nucleotides in length. 
     
     
         30 . The system of  claim 27 , wherein the molecular beacon probe homopolymeric nucleotide sequence of 10-15 repeated nucleotides consist of: homopolymeric A;
 homopolymeric G; homopolymeric T; or homopolymeric C.   
     
     
         31 . The system according to  claim 27 , wherein the cartridge comprises at least two molecular beacon probes, wherein each of the at least two molecular beacon probes comprises a different homopolymeric nucleotide sequence. 
     
     
         32 . The system according to  claim 27 , wherein the different homopolymeric nucleotide sequences differ in length and/or homopolymeric sequence. 
     
     
         33 . The system according to  claim 27 , comprising a plurality of molecular beacon probes, wherein each of the plurality of molecular beacon probe comprises non-homopolymeric sequence complementary to the target nucleic acid; and wherein each of the plurality of molecular beacon probes comprises different non-homopolymeric flanking sequences. 
     
     
         34 . The system according to  claim 27 , wherein the cartridge is engaged with the device at the cartridge receiver. 
     
     
         35 . The system according to  claim 27 , wherein the software is programmed to calculate a melting peak as the negative values of the first derivatives of the obtained melting curves.