Detection of short homopolymeric repeats
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. A method for deducing the number of nucleotides present in a target homopolymeric repeat sequence equal to or shorter than 15bp in length, the method comprising the steps of:
a) generating amplicons by amplifying a nucleic acid sequence comprising the target homopolymeric repeat sequence;
b) heating the generated amplicons in the presence of at least one signal-generating oligonucleotide probe comprising a sequence capable of hybridizing to the target homopolymeric repeat sequence, and detecting the changes in the strength of the signal generated by said probe in the function of temperature to obtain at least one melting curve; and
c) deducing the number of nucleotides present in the target homopolymeric repeat sequence from the at least one melting curve;
wherein the sequence capable of hybridizing to the target homopolymeric repeat sequence comprises a sequence identical to or complementary to a mutant sequence comprising a deletion of at least one homonucleotide in said target homopolymeric repeat sequence, and
wherein the step of generating amplicons is performed in a PCR comprising a polymerase having 3′-5′ exonuclease activity.
2. The method according to claim 1 , wherein the oligonucleotide probe is a molecular beacon oligonucleotide probe.
3. The method according to claim 2 , wherein at least a second molecular beacon oligonucleotide probe differently labeled than the first molecular beacon oligonucleotide probe is used, wherein said second molecular beacon oligonucleotide probe comprises a sequence capable of hybridizing to a second target homopolymeric nucleotide repeat sequence being different from the first target homopolymeric nucleotide repeat sequence.
4. The method according to claim 3 , wherein the sequence capable of hybridizing to the second target homopolymeric nucleotide repeat sequence comprises a sequence identical to or complementary to a mutant sequence comprising a deletion of at least one homonucleotide in said second target homopolymeric repeat sequence.
5. The method according to claim 1 , wherein the at least one signal-generating labeled oligonucleotide probe comprises a structural feature or modification protecting said probe from the 3′-5′ exonuclease activity of the polymerase.
6. The method according to claim 5 , wherein the sequence- specific probe is a molecular beacon oligonucleotide probe and wherein said structural feature or modification is a 3′-5′exonuclease-activity resistant stem.
7. The method according to claim 5 , wherein the structural feature or modification is selected from:
i) inverted dT at the 3′ end of the probe;
ii) at least one phosphorothioate linkage positioned before any of the last three nucleotides at the 3′ end of the probe.
8. The method according to claim 1 , wherein the steps a), b), and c) are performed in an automated system.
9. The method according to claim 8 , said method preceded by any of the following steps:
providing a source of a nucleic acid potentially comprising the target homopolymeric repeat sequence;
liberating and/or isolating the nucleic acid potentially comprising the target homopolymeric repeat sequence from the source of a nucleic acid; and
providing said liberated and/or purified nucleic acid potentially comprising the target homopolymeric repeat sequence to the step of generating amplicons; and
wherein at least the steps of:
liberating and/or isolating the nucleic acid potentially comprising the target homopolymeric repeat sequence from the source of a nucleic acid;
providing said liberated and/or purified nucleic acid potentially comprising the target homopolymeric repeat sequence to the step of generating amplicons;
are also performed in an automated system.
10. The method according to claim 9 , wherein at least the steps of:
liberating and/or isolating the nucleic acid potentially comprising the target homopolymeric repeat sequence from the source of a nucleic acid;
providing said liberated and/or purified nucleic acid potentially comprising the target homopolymeric repeat sequence to the step of generating amplicons;
generating amplicons by amplifying a nucleic acid sequence comprising the target homopolymeric repeat sequence; and
heating the generated amplicons in the presence of a signal-generating oligonucleotide probe and detecting the changes in the strength of said signal in the function of temperature to obtain at least one melting curve;
are performed in a cartridge engageable with said automated system.
11. The method according to claim 9 , wherein the source of a nucleic acid is a biological sample.
12. A method according to claim 1 , wherein the deducing the number of nucleotides present in the target homopolymeric repeat sequence from the at least one melting curve is performed by assessing the position or a relative position of at least one peak of the first derivative of said melting curve.
13. The method according to claim 12 , wherein said method is performed in an automated manner.