Determination of the number of tandem repeat nucleotides using encoded probe-displaying beads
View Patent ↗This invention provides compositions and methods for genetic testing of an organism and for correlating the results of the genetic testing with a unique marker that unambiguously identifies the organism. The markers may be internal markers, such as for example single nucleotide polymorphisms (SNPs), short tandem repeats (STRs), or other sites within a genomic locus. Alternatively, the markers may be external, such that they are separately added to the genetic sample before testing.
1. A method of determining the number of tandem nucleotide repeats in a target nucleic acid sequence, wherein said tandem repeats are flanked at each side by a non-repeat flanking sequence, the method comprising the following steps:
(a) providing a set of oligonucleotide probes attached to beads, said beads being associated with a chemically or physically distinguishable characteristic that uniquely identifies the probes attached thereto, said probes capable of annealing to the target nucleic acid sequence and containing an interrogation site, wherein probes in the set differ in a the number of contiguous repeated subsequences they contain, such that when the probes are annealed to the target sequence, the interrogation sites of probes are aligned with a target site which is located within the repeated subsequences or not;
(b) contacting a target sequence to said probes, under conditions so that said target sequence forms hybridization complexes with said probes;
(c) interrogating the hybridization complexes between the target sequence and the probes, to determine which probes have their interrogation sites outside the repeated subsequences and which probes do not; and
(d) decoding the set of probes using the distinguishable characteristic of the beads, and based on which probes do and do not have their interrogation sites outside the repeated subsequences, such that the number of contiguous repeated subsequences in probes in hybridization complexes which have their interrogation sites outside the repeated subsequences in the targets represent the number of repeats in the target sequence.
2. The method of claim 1 , wherein step (c) comprises contacting the hybridization complexes with a sequence-specific polymerase and labeled nucleotide triphosphates, such that the nucleotide triphosphates are incorporated into one or more probes thereby forming elongated probes, and wherein the labels in the elongated probes indicate whether the interrogation sites align outside the repeated subsequences and which do not.
3. The method of claim 1 , wherein the beads are arranged in a planar assembly and the characteristic is optically detectable by direct imaging.
4. The method of claim 1 wherein the target nucleic acid sequence is derived from a patient genomic sample by amplification of one or more nucleic acid subsequences within the patient genomic sample.
5. The method of claim 4 , wherein the amplification is performed using the polymerase chain reaction.
6. The method of claim 1 wherein the oligonucleotide probes are capable of being elongated from the interrogation site following hybridization, by incorporation of one or more types of deoxyribonucleotide triphosphates or di-deoxyribonucleotide triphosphates.
7. The method of claim 6 wherein at least one type of deoxyribonucleotide triphosphate or di-deoxyribonucleotide triphosphate is labeled so as to generate an optically detectable signature associated with the elongation product following incorporation.
8. The method of claim 7 wherein the optically-detectable labels are fluorophore dyes.
9. The method of claim 7 wherein the label is an inorganic nanoparticle label.
10. The method of claim 9 wherein the nanoparticle label is semiconductor Quantum Dot particles or Resonant Light Scattering metal nanoparticles.