Nucleic acid testing method for point-of-care diagnostics and genetic self-monitoring

• US 20080050735A1
• Filed: 02/01/2007
• Published: 02/28/2008
• Est. Priority Date: 02/01/2006
• Status: Abandoned Application

• Current Assignee: Elena Pushnova
• Original Assignee: Elena Pushnova
• Inventors: Pushnova, Elena
• Application Number:
  US11/702,028
• Publication Number:
  US 20080050735A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein

Abstract

This invention describes a nucleic acid testing procedure in a form of portable device or a test kit for the purposes of clinical genetic testing, infectious disease diagnostics, biodefense, forensic analysis, paternity testing, pet and cattle breeding, food testing, etc. This testing does not include toxic chemicals and is simple enough to be used by an average individual without any special laboratory training. The procedure includes collecting the sample, potential isothermal amplification of the whole genomic DNA or a fragment of genomic DNA, denaturing double-stranded DNA into single-stranded form, hybridizing the denatured sample DNA to single-stranded allele-specific tester oligonucleotides complementary to the analyzed DNA sequence of interest, selective removal of single-stranded DNA from DNA hybrids, and finally detecting the label in double-stranded hybrids to determine the presence or absence of a particular sequence in the initial sample.

Claims

1. A genetic testing procedure for identification of DNA sequence(s) of interest in the sample that is carried out within a field device or in a test kit format, that is applicable for self-executed diagnostics by any individual, without laboratory settings, toxic chemicals or specific skills being required, and is comprised of:

a) Denaturing double-stranded DNA in the analyzed sample to single-stranded form, where depending on availability of DNA in the sample, genomic DNA may be partially or fully amplified prior to denaturing, and where RNA may be analysed in the same manner as DNA with unstable RNA being converted to DNA by reverse transcription prior to denaturing, and where positive and negative control DNA fragments may be included in the test.

b) Hybridizing denatured DNA with single-stranded sequence-specific or allele-specific tester oligonucleotides complementary to the DNA sequence of interest, where depending on the type of the test, one (e.g. for detection of infectious agents) or more (for diploid or polyploid genotype diagnostics, multiplex genotyping, detection of multiple infectious agents, DNA fingerprinting, paternity testing, etc.) tester oligonucleotides may be used, and where the end nucleotide at either 3′

- or 5′

-end of the tester oligonucleotide is associated with a label;

where if two or more tester oligonucleotides are used in the test, the end nucleotide of each sequence-specific or allele-specific tester oligonucleotide is complimentary to a particular polymorphic nucleotide characteristic for each variant or allele of the sequence of interest and is associated with a label which is unique to each sequence or allele, and where the opposite end of each tester oligonucleotide is modified to allow capture of the oligonucleotide;

where in addition to label associated with the end sequence-specific or allele-specific nucleotide of the tester oligonucleotide and therefore called a detector label, a different control label may be attached to the nucleotide immediately adjacent to the end detector nucleotide and complimentary to any variant or allele of the sequence of interest in order to control successful hybridization between single-stranded sample DNA and the tester oligonucleotide; and

where positive and negative control tester oligonucleotides complimentary to sequences that are known to be present and absent in the sample, respectively, may be included into the test.

c) Subjecting double-stranded DNA hybrids and possible excess of single-stranded tester oligonucleotides and single-stranded sample DNA to treatment with an agent that cleaves mispaired nucleotides from nucleic acid strand to digest any excess of tester oligonucleotides and single-stranded sample DNA and to remove the mismatched labeled end detector nucleotides from double-stranded hybrids;

where properly hybridized end detector nucleotides and control nucleotides are not removed from double-stranded hybrids.

d) Capturing double-stranded hybrids on a carrier.

e) Registering the label present in the captured DNA hybrids, where the presence of a particular sequence-specific or allele-specific label in the captured hybrids indicates the presence of the corresponding sequence or allele in the analyzed sample, and where the presence of the control label indicates successful hybridization between single-stranded sample DNA and the tester oligonucleotide and therefore confirms validity of the result.

2. The procedure of claim 1, wherein the opposite-to-label ends of tester oligonucleotides do not have modifications for oligonucleotide capturing, and in step d) the double-stranded DNA hybrids are separated from free nucleotides after cleavage step c) in a size-separation medium;

and wherein the presence of particular label in the higher molecular weight double-stranded DNA hybrid fraction in step e) indicates the presence of the corresponding sequence or allele in the analyzed sample.

3. The procedure of claim 2, wherein sequence-specific or allele-specific tester oligonucleotides have different lengths but have the same label associated with all sequence-specific or allele-specific end nucleotides;

and wherein after separating the double-stranded DNA hybrids in a size-separation medium the presence of a particular sequence or allele in the analyzed sample is determined in step e) by presence of the label in a corresponding sequence-specific or allele-specific hybrid size fraction.

4. The procedure of claim 1, wherein hybridization with each sequence-specific or allele-specific tester oligonucleotide and capturing of double-stranded hybrids is performed in a separate reaction, and the end nucleotides of all sequence-specific or allele-specific tester oligonucleotides have the same label;

and wherein the presence of the label in a captured hybrid in a particular sequence-specific or allele-specific reaction determined as in step e) indicates the presence of the corresponding sequence or allele in the analyzed sample.

5. The procedure of claim 1, wherein the labeled tester oligonucleotides are captured on the carrier prior to hybridization with denatured sample DNA.

6. The procedure of claim 5, wherein each sequence-specific or allele-specific tester oligonucleotide is attached to a separate carrier, and end nucleotides of all tester oligonucleotides have the same label;

and wherein the presence of the label in a particular sequence-specific or allele-specific carrier indicates the presence of the corresponding sequence or allele in the analyzed sample.

7. The procedure of claim 5, wherein each sequence-specific or allele-specific tester oligonucleotide is attached to a designated spot on the same carrier, and end nucleotides of all tester oligonucleotides have the same label;

and wherein the presence of the label in a particular sequence-specific or allele-specific spot indicates the presence of the corresponding sequence or allele in the analyzed sample.