US5888733A — In situ hybridization to detect specific nucleic …

Claim 1

Independent Claim 165 words

A method for detecting the presence of specific RNA sequences in a sample of eukaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, during which step the sample is subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be detected; (3) removing any unbound and any non-specifically bound binding partner; (4) detecting the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Claim 2

Independent Claim 165 words

A method for detecting the presence of specific RNA sequences in a sample of eukaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, during which step the sample is subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be detected; (3) removing any unbound and any non-specifically bound binding partner; (4) detecting the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Claim 3

Independent Claim 171 words

A method for detecting the presence of specific RNA sequences in a sample of a eukaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, during which step the sample is subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount greater than 10% and effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be detected, (3) removing any unbound and any non-specifically bound binding partner, and (4) detecting the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Claim 4

Independent Claim 171 words

A method for detecting the presence of specific RNA sequences in a sample of a eukaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, during which step the sample is subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount greater than 10% and effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be detected, (3) removing any unbound and any non-specifically bound binding partner, and (4) detecting the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Claim 5

Dependent Claim 143 words

A method according to claim 1 or 2, wherein the polymeric strand comprises polymerized moieties of formula (I) ##STR7## wherein Y designates O or S, each X independently designates O or S, each Q designates a ligand that independently is a naturally occurring nucleobase, a non-naturally occurring nucleobase, an intercalator, a nucleobase-binding group, a label or H, u is an integer from 1 to 5, p and s independently designate 0 or 1, q and r independently designate 0 or 1, t designates 0 or 1, R 1 and R 10 independently designate H or C 1-4 alkyl, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 independently designate H, the side chain of a naturally occurring amino acid or the side chain of a non-naturally occurring amino acid.

Claim 6

Dependent Claim 81 words

A method according to claim 3, wherein u, p, q, r, s, Y, X and Q are as defined in claim 2, t is 0, R 1 designates H or CH 3 , R 3 , R 4 , R 6 and R 9 designate H, and R 2 , R 5 , R 7 and R 8 independently designate H or the side chain of a naturally occurring amino acid or the side chain of a non-naturally occurring amino acid.

Claim 7

Dependent Claim 92 words

A method according to claim 3, wherein the polymeric strand comprises polymerized moieties of formula (II), which are moieties of the general formula (I) wherein r is 0 and q and s are 1, ##STR8## wherein Y, X, Q, p, t and u are as defined in claim 2, R 2 , R 5 and R 7 independently designate H, the side chain of a naturally occurring amino acid or the side chain of a non-naturally occurring amino acid, and R 1 and R 10 independently designate H or CH 3 .

Claim 8

Dependent Claim 80 words

A method according to claim 3, wherein the polymeric strand comprises polymerized moieties of formula (III), which are moieties of the general formula (I) wherein p, r and t are 0 and q and s are 1 ##STR9## wherein Y, X, Q and u are as defined in claim 2, R 2 , R 5 and R 7 independently designate H, the side chain of a naturally occurring amino acid or the side chain of a non-naturally occurring amino acid.

Claim 9

Dependent Claim 88 words

A method according to claim 3, wherein the polymeric strand comprises polymerized moieties of formulas (IV)-(VI), which are moieties of the general formula (I) wherein p, r and t are 0 and u, s and q are 1, ##STR10## wherein R 2 , R 5 and R 7 designate H, the side chain of a naturally occurring amino acid or the side chain of a non-naturally occurring amino acid, and each Q independently designates a naturally occurring nucleobase, a non-naturally occurring nucleobase, an intercalator or a nucleobase-binding group.

Claim 10

Dependent Claim 43 words

A method according to claim 7, wherein R 2 , R 5 and R 7 designate H or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q designates thymine, adenine, cytosine, guanine, uracil or hypoxanthine.

Claim 11

Dependent Claim 46 words

A method according to claim 3, wherein the polymeric strand comprises polymerized moieties of formula (VII), which are moieties of formula (I) wherein p, r and t are 0 and u, sand q are 1, ##STR11## wherein Q designates thymine, adenine, cytosine, guanine, uracil or hypoxanthine.

Claim 12

Dependent Claim 24 words

A method according to claim 3, wherein the polymeric strand is made up of from 8 to 30 polymerized moieties of the formula (I)-(VII).

Claim 13

Dependent Claim 22 words

A method according to claim 1, wherein polymerized moieties of formulas (IV)-(VII) constitute at least 75% by weight of the polymeric strand.

Claim 14

Dependent Claim 17 words

A method according to claim 1, wherein the concentration of the hybrid destabilizing agent is above 10%.

Claim 15

Dependent Claim 25 words

A method according to claim 12 or 2, wherein the hybrid destabilizing agent is selected from the group consisting of formamide, ethylene glycol and glycerol.

Claim 16

Dependent Claim 20 words

A method according to claim 13, wherein the hybrid destabilizing agent is formamide in a concentration from 30% to 50%.

Claim 17

Dependent Claim 21 words

A method according to claim 13, wherein the hybrid destabilizing agent is ethylene glycol in a concentration from 50% to 65%.

Claim 18

Dependent Claim 29 words

A method according to claim 1 or 2, wherein the sample of eucaryotic origin is a tissue section, a cell smear, or a suspension of cells or parts thereof.

Claim 19

Dependent Claim 22 words

A method according to claim 1, wherein the binding partner further comprises one or more labels which may be identical or different.

Claim 20

Dependent Claim 30 words

A method according to claim 17, wherein the label is selected from the group consisting of fluorescent labels, biotin, digoxigenin, dinitro benzoic acid, peptide labels, rhodamine, R-phycoerythrine and cyanine dyes.

Claim 21

Dependent Claim 15 words

A method according to claim 18, wherein at least one label is a fluorescent label.

Claim 22

Dependent Claim 26 words

A method according to claim 19, wherein the unbound and non-specifically bound binding partner in step (3) is removed using a washing buffer at alkaline pH.

Claim 23

Dependent Claim 22 words

A method according to claim 20, wherein the washing buffer in step (3) has a pH value of from 8 to 10.5.

Claim 24

Dependent Claim 15 words

The method according to claim 1, wherein said RNA is mRNA, rRNA or nuclear RNA.

Claim 25

Independent Claim 170 words

A method for determining the presence of specific RNA sequences in a sample of a eucaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, whereby the sample will be subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing from 8 to 30 polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be determined, (3) removing any unbound and any non-specifically bound binding partner, and (4) determining the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Claim 26

Independent Claim 170 words

A method for determining the presence of specific RNA sequences in a sample of a eucaryotic origin using in situ hybridization which method comprises the steps of (1) producing a preparation of said sample, whereby the sample will be subject to fixation, (2) contacting said preparation with a hybridization solution comprising at least one binding partner capable of hybridizing to a specific RNA sequence to be detected so as to form hybrids and a hybrid destabilizing agent in an amount effective to decrease the melting temperature of hybrids formed between said RNA and said binding partner so as to increase the ratio between specific binding and non-specific binding, said binding partner being a polymeric strand containing from 8 to 30 polymerized moieties having a non-cyclic backbone, the polymeric strand being capable of hybridizing to the RNA sequence to be determined, (3) removing any unbound and any non-specifically bound binding partner, and (4) determining the presence of bound binding partner in the preparation thereby detecting the presence of specific RNA sequences.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2002-03-29	2002-09-28	2003-03-30	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2006-03-29	2006-09-28	2007-03-30	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2010-03-29	2010-09-28	2011-03-30	$7700.00	Window Closed / Lapsed

In situ hybridization to detect specific nucleic acid sequences in eucaryotic samples

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