In-situ methods for analyzing target sequences using linear beacons

• US 6,649,349 B2
• Filed: 09/10/2001
• Issued: 11/18/2003
• Est. Priority Date: 10/27/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Coull, James M., Gildea, Brian D., Hyldig-Nielsen, Jens J.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/950,459
• Publication Number:
  US 20020064772A1
• Time in Patent Office: 799 Days
• Field of Search: 435/5, 435/6, 436/94
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention is directed to methods, kits and compositions pertaining to Linear Beacons. In the absence of a target sequence, Linear Beacons facilitate efficient energy transfer between the donor and acceptor moieties linked to opposite ends of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.

Claims

1. A method for in-situ analysis of a target sequence of an organism of interest in a sample, said method comprising:

a) providing a sample of fixed cells for in-situ analysis;

b) treating the sample with a polymer comprising at least one linked energy donor moiety and at least one linked energy acceptor moiety wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution, is substantially independent of at least two variables selected from the group consisting of;

i) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

ii) spectral overlap of the at least one linked energy donor moiety and the at least one linked energy acceptor moiety;

iii) presence or absence of magnesium in the aqueous solution; and

the

iv) ionic strength of the aqueous solution;

c) detecting, identifying or quantitating the hybridization of the polymer to the target sequence, under suitable in-situ hybridization conditions, wherein the presence, absence or amount of target sequence present in the sample is correlated with a change in detectable signal associated with at least one donor or acceptor moiety of the polymer; and

d) detecting, identifying or quantitating the organism of interest in the sample based upon the presence, absence or amount of the hybridization of the polymer to the target sequence that is determined.

2. The method of claim 1, wherein excess polymer is not washed away prior to performing step (c).

3. The method of claim 1, wherein blocker probes are added to improve assay performance.

4. The method of claim 1, further comprising:

e) treating, before performing steps c) or d), the sample with at least one additional independently detectable polymer comprising at least one linked energy donor moiety and at least one linked energy acceptor moiety wherein said donor and acceptor moieties are separated by at least a portion of a probing nucleobase sequence and wherein said polymer does not form a stem and loop hairpin and is further characterized in that the efficiency of transfer of energy between said donor and acceptor moieties, when the polymer is solvated in aqueous solution, is substantially independent of at least two variables selected from the group consisting of;

i) nucleobase sequence length separating the at least one energy donor moiety from the at least one energy acceptor moiety;

ii) spectral overlap of the at least one linked energy donor moiety and the at least one linked energy acceptor moiety;

iii) presence or absence of magnesium in the aqueous solution; and

the

iv) ionic strength of the aqueous solution; and

f) performing steps c) and d) in multiplex mode such that the independently detectable signal from each of the polymers is selected to independently detect, identify or quantitate two or more different organisms of interest in the sample.

5. The method of claim 1, wherein the determination of detectable signal is made using a microscope, laser scanning device or flow cytometer.

6. The method of claim 1, wherein the organism of interest is a bacteria or a virus.

7. The method of claim 1, wherein the method is repeatedly performed to determine the effect of antimicrobial agents on the growth of one or more microorganisms.

8. The method of claim 1, wherein the method is selected to determine target sequences that are particular to organisms found in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples.

9. A method for in-situ analysis of a target sequence of an organism of interest in a sample, said method comprising:

a) providing a sample of fixed cells for in-situ analysis;

b) treating the sample with a polymer comprising;

i) a probing nucleobase sequence for probing a target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

ii) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

iii) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence;

c) detecting, identifying or quantitating the hybridization of the polymer to the target sequence, under suitable in-situ hybridization conditions, wherein the presence, absence or amount of target sequence present in the sample is correlated with a change in detectable signal associated with at least one donor or acceptor moiety of the polymer; and

d) detecting, identifying or quantitating the organism of interest in the sample based upon the presence, absence or amount of the hybridization of the polymer to the target sequence that is determined.

10. The method of claim 9, wherein excess polymer is not washed away prior to performing step (c).

11. The method of claim 9, wherein blocker probes are added to improve assay performance.

12. The method of claim 9, further comprising:

e) treating, before performing steps c) or d), the sample with at least one additional independently detectable polymer comprising;

i) a probing nucleobase sequence for probing a target sequence to which the probing nucleobase sequence is complementary or substantially complementary;

ii) at least one energy donor moiety that is linked to the probing nucleobase sequence; and

iii) at least one energy acceptor moiety that is linked to the probing nucleobase sequence wherein the at least one donor moiety is separated from the at least one acceptor moiety by at least a portion of the probing nucleobase sequence; and

f) performing steps c) and d) in multiplex mode such that the independently detectable signal from each of the polymers is selected to independently detect, identify or quantitate two or more different organisms of interest in the sample.

13. The method of claim 9, wherein the determination of detectable signal is made using a microscope, laser scanning device or flow cytometer.

14. The method of claim 9, wherein the organism of interest is a bacteria or a virus.

15. The method of claim 9, wherein the method is repeatedly performed to determine the effect of antimicrobial agents on the growth of one or more microorganisms.

16. The method of claim 9, wherein the method is selected to determine target sequences that are particular to organisms found in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples.