US 2011 01961.46A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0196146A1 KHRPN et al. (43) Pub. Date: Aug. 11, 2011

(54) METHODS, COMPOSITIONS, AND KITS FOR Publication Classification RECOVERY OF NUCLECACDS OR PROTEINS FROM FIXED TISSUE SAMPLES (51) Int. Cl. C7H I/06 (2006.01) (75) Inventors: Yuri KHRIPIN, Gaithersburg, MD CI2N 9/00 (2006.01) (US); Arvind Virmani, Gaithersburg, MD (US); Lori Kobayashi, Boonsboro, MD (US) (52) U.S. Cl...... 536/25.41; 435/270; 435/183 (73) Assignee: QIAGEN GAITHERSBURG INC., GAITHERSBURG, MD (US) (57) ABSTRACT (21) Appl. No.: 12/984,391 Methods and materials for improving nucleic acid or protein recovery from samples preserved in liquid cytological preser (22) Filed: Jan. 4, 2011 Vative solutions by utilizing scavenging agents, such as hydrazine- and hydrazide-containing compounds, are pro Related U.S. Application Data vided. Lysis solutions comprising hydrazine- and hydrazide (60) Provisional application No. 61/292,078, filed on Jan. containing compounds and kits comprising the same are also 4, 2010. provided.

NH PH MI - - NH H H

h" ',H | ar"H OH aH - byH H'hH Aminoguanidine Fomaldehyde

NH H NH H -H,0 HN : ... H HN N H Methylenaring articline Patent Application Publication Aug. 11, 2011 Sheet 1 of 9 US 2011/O1961.46 A1

Aminoguanidine Formaldehyde

H.0 NH H H NH-H... ,H Hs NH -----|--|--Ho "?" . . H "" ; ; ; HN N Methylenaminoguanidire

Fig. 1 Patent Application Publication Aug. 11, 2011 Sheet 2 of 9 US 2011/O1961.46 A1

no scovenger scovenger Patent Application Publication Aug. 11, 2011 Sheet 3 of 9 US 2011/O1961.46 A1

s 20,000 SiHa

Water O.6M 0.3M OSM Concentration of Succinic Acid Dihydrazide Fig. 3 Patent Application Publication Aug. 11, 2011 Sheet 4 of 9 US 2011/O1961.46 A1

Sample Water Concentration of Adipic Acid Dihydrazide Fig. 4 Patent Application Publication Aug. 11, 2011 Sheet 5 of 9 US 2011/O1961.46 A1

Water

scavenger

O 2.5K SK OK 2OK Number of SiHa cells Fig. 5 Patent Application Publication Aug. 11, 2011 Sheet 6 of 9 US 2011/O1961.46 A1

--no pre-lysis -- d minute pre-lysis

wder 0.3M Adipicocid (,5M Aninoguanidine Scavenger

Fig. 6 Patent Application Publication Aug. 11, 2011 Sheet 7 of 9 US 2011/O1961.46 A1

E10 rhinute incubation overnight incubation

Water Adipic Succinic AminoGuantidine Scavenger Fig. 7 Patent Application Publication Aug. 11, 2011 Sheet 8 of 9 US 2011/O1961.46 A1

2O,OOOSa

Water O.3M O.3M Oinute O.3M Orinite incubation: W fo PK incubation WAPK Condition

Fig. 8 Patent Application Publication Aug. 11, 2011 Sheet 9 of 9 US 2011/O1961.46 A1

--Handyabs RT elution -E-Qlogen RT elution A-Qiagen heated elution

WCter O,3M Adipicocid O.3M Succinic Cucid Scavenger

Fig. 9 US 2011/01961.46 A1 Aug. 11, 2011

METHODS, COMPOSITIONS, AND KITS FOR are prepared for electron microscopy. Potassium dichromate, RECOVERY OF NUCLECACDS OR chromic acid, and potassium permanganate are also used in PROTEINS FROM FIXED TISSUE SAMPLES specific histological preparations. Two common precipitating fixatives are ethanol and methanol. Acetone is also used. REFERENCE TO RELATED APPLICATIONS 0010 Acetic acid is a denaturant that is sometimes used in 0001. This application claims priority to U.S. Provisional combination with other precipitating fixatives. Alcohols, by themselves, are known to cause shrinkage of tissue during Patent Application No. 61/292,078, filed on Jan. 4, 2010, fixation while acetic acid alone is associated with tissue which is incorporated herein by reference in its entirety. Swelling; combining the two may result in better preservation FIELD OF THE INVENTION of tissue morphology. Other fixative agents include picric acid and mercuric chloride. 0002 Methods and kits for improving recovery of nucleic 0011. One of the problems with fixing biological samples acids or proteins that have been fixed in tissue or cell Samples is that the nucleic acids and proteins in the samples may be are described. irreversibly bound to the fixative agent(s). Even if the nucleic acids and proteins are not irreversibly bound to the fixative BACKGROUND OF THE INVENTION agent(s), removal of excess fixative agents from the samples 0003. In the fields of histology, pathology, and cell biol may be important for reliable recovery and analysis of nucleic ogy, fixation is a chemical process by which biological acids and proteins. Additionally, fixative agents may interfere samples are preserved from decay. Fixation terminates any with the use of the isolated protein or nucleic acid in down ongoing biochemical reactions, and may also increase the stream biochemical analyses, such as PCR. mechanical strength or stability of the treated samples. The purpose of fixation is to preserve a sample of biological SUMMARY OF THE INVENTION material as close to its natural state as possible. Fixed samples are used for examination or analysis. 0012. The present disclosure relates to methods of recov 0004 Immersion is a fixation technique in which the ering and analyzing a target molecule, such as a nucleic acid sample is immersed in fixative of Volume at a minimum of 20 or protein, from a fixed biological sample and materials and times greater than the volume of the tissue to be fixed. The kits useful in Such methods. fixative diffuses through the tissue in order to fix, so tissue 0013. In one embodiment, a method is provided for size and density, as well as the type of fixative must be taken extracting a target molecule from a biological sample pre into account. Using a larger sample means it will take longer served in a liquid cytology preservative solution. for the fixative to reach the deeper tissue. 0014. In an embodiment, the method comprising: A) con 0005 Fixative agents can be classified as crosslinking or tacting the biological sample with a scavenger solution com precipitative fixatives. Crosslinking fixatives act by creating prising a scavenging agent comprising at least one terminal covalent chemical bonds between proteins in tissue. This hydrazine group of the formula: anchors soluble proteins to the cytoskeleton, and lends addi tional rigidity to the tissue. Precipitating, or denaturing, fixa tives act by reducing the solubility of protein molecules and, often, by disrupting the hydrophobic interactions which give many proteins their tertiary structure. 0006. One commonly used fixative in histology is the and crosslinking fixative formaldehyde, which is often sold as a B) treating the biological sample under conditions sufficient saturated aqueous Solution under the name formalin. Form to release the nucleic acid or protein from the biological aldehyde is thought to interact primarily with the residues of sample; and C) recovering the target molecule from the iso the basic amino acid lysine. lation Solution, wherein said target molecule is a nucleic acid 0007 Another popular aldehyde for fixation is glutaralde or a polypeptide. hyde, which is believed to operate by a similar mechanism to 0015. In one embodiment, the scavenging agent is selected formaldehyde. from the group consisting of 0008 Formaldehyde preserves or fixes tissue or cells by a) a compound according to formula I: cross-linking primary amino groups in proteins or nucleic acids through a —CH2— linkage, i.e., a methylene bridge. Because formaldehyde is highly reactive, excessive formal (I) dehyde in the sample or media interferes with any sample N processing or analysis that involves functional proteins (such N- ma-NH2:H as enzymes or antibodies), nucleic acid probes, resins, or any NH other functional reagents with amino groups by cross-linking to these amino groups with Subsequent reagent deactivation. Moreover, since the cross-links can be reversed by heat, any and excessive formaldehyde in the media will eventually form b) a compound according to formula II: cross links again, preventing cross-link reversal from being effective. 0009. Oxidizing fixatives can react with various side (II) chains of proteins and other biomolecules, allowing the for R If(R-N-NH), P 2-N- mation of crosslinks which stabilize tissue structure. Osmium tetroxide is often used as a secondary fixative when samples US 2011/01961.46 A1 Aug. 11, 2011 wherein R' is selected from the group consisting of: C-C2 0024. In another embodiment, the biological sample is alkyl, C-C alkenyl: C-C cycloalkyl, C-C cycloalkenyl: contacted with the Scavenger Solution before the target mol Co-Co aryl; and Co-Co heteroaryl; R, which in each ecule is released from the biological sample. instance may be the same or different, is selected from the 0025. In another embodiment, the target molecule is group consisting of released from the biological sample by lysing the biological sample in the presence of a lysis solution. 0026. In another embodiment, the scavenger solution (i) functions as the lysis solution. 0027. In another embodiment, the scavenger solution is added to biological sample before, after, or at the same time as (ii) the lysis solution. 0028. In another embodiment, the target molecule is a target nucleic acid and the target nucleic acid is recovered from the scavenger Solution by a method comprising: (i) hybridizing a nucleic acid probe to the target nucleic acid with (iii) a second nucleic acid to form a nucleic acid hybrid; (ii) binding the nucleic acid hybrid to a solid phase; (iii) separat ing the solid phase from the scavenger Solution; and (iv) eluting the target nucleic acid from the Solid phase. 0029. In another embodiment, the nucleic acid hybrid is a m is an integer selected from the group consisting of 0 and 1: DNA:RNA hybrid. and n is an integer selected from the group consisting of 1 and 0030. In another embodiment, the nucleic acid hybrid is 2. In certain embodiments, the Scavenging agent may be bound to the Solid phase by a method comprising contacting modified by methods known in the art to increase the solubil the nucleic acid hybrid with an antibody capable of binding to ity of the scavenging agent in water. For example, R' option the nucleic acid hybrid, wherein the antibody is bound to the ally may be substituted with constituents that increase the solid phase or adapted to be bound to the solid phase. hydrophilicity of the R' constituent. 0031. In another embodiment, the target nucleic acid is 0016. In another embodiment, the Scavenging agent is a eluted from the solid phase at an elution temperature of from compound of formula I wherein mis 1 and R' is selected from about 20° C. to about 70° C. the group consisting of C-C alkyl, C-C alkyl, and C-C, 0032. In another embodiment, the target nucleic acid is alkyl. eluted from the solid phase at an elution temperature of from 0.017. In another embodiment, the Scavenging agent is a about 50° C. to about 60° C. compound of formula II whereinn is 2, R is selected from the 0033. In another embodiment, alysis solution is provided, group consisting of C-C alkyl, C-C alkyl, and C-C, comprising: alkyl; and R is 0034 (i) a buffer; 0035 (ii) a detergent; 0036 (iii) a Scavenging agent comprising at least one O terminal hydrazine group of the formula - C H 0.018. In another embodiment, the scavenging agent is Jwn N-NH2: selected from the group consisting of semicarbazide; thi osemicarbazide; carbazide; thiocarbazide, N-aminoguani and dine and a salt thereof, including hydrochloride salts; N.N- 0037 (iv) optionally, protein digestive enzyme. diaminoguanidine and a salt thereof, including 0038. In another embodiment, the scavenging agent of the dihydrochloride salts; acetylhydrazide; adipic acid dihy lysis Solution is selected from the group consisting of: drazide. Succinic acid dihydrazide; formic hydrazide; maleic 0039 a) a compound according to formula I: acid dihydrazide; malonic acid dihydrazide; benzenesulfo nylhydrazide; tosylhydrazide; methylsulfonylhydrazide. 0019. In another embodiment, the scavenger solution NH (RI) (I) comprises from about 0.1M to about 1.OM, from about 0.1M 2 iii. NH2: to about 0.5M, from about 0.2M to about 0.4M, or about 0.3M r"H of the Scavenging agent. NH 0020. In another embodiment, about 0.3M adipic acid dihydrazide or about 0.3M Succinic acid dihydrazide is used. 0021. In another embodiment, the scavenger solution is and added directly to the liquid cytology preservative solution. 0040 b) a compound according to formula II: 0022. In another embodiment, the scavenger solution comprises about 2 parts by Volume of the lysis solution and about 1 part by volume of the biological sample preserved in the liquid cytology preservative solution. 0023. In another embodiment, the scavenger solution fur ther comprises a protein digestive enzyme. US 2011/01961.46 A1 Aug. 11, 2011

wherein: concentration, while the left bar represents the result of add I0041) R' is selected from the group consisting of: ing an equal Volume of water. The results of duplicate experi C-C alkyl, C-C alkenyl: C-C cycloalkyl, C-C, ments are shown. cycloalkenyl: C-C aryl; and C-Co heteroaryl, 0.052 FIG. 3 demonstrates the effect of varying succinic wherein R' is optionally substituted so as to increase the acid dihydrazide concentration. Solubility of the scavenging agent in water, 0053 FIG. 4 demonstrates the effect of varying the con I0042) R' which in each instance may be the same or centration of adipic acid dihydrazide on samples in SurePath different, and is selected from the group consisting of: medium. For each condition, the left bar represents data in the absence of cells, while the right bar represents data obtained with 10,000 SiHa cells.“Sample” indicates SurePath medium (i) alone. “0.16M and “0.30M indicate that the lysis buffer O comprising adipic acid dihydrazide was added to the Sure -C- Path sample to a final concentration of 0.16M and 0.30M, (ii) respectively. “Water indicates that water was used in place of O SurePath. -- and 0054 FIG.5 demonstrates the effectiveness of adipic acid dihydrazide as a scavenger when the cell number is varied. O 0055 FIG. 6 illustrates the effect of a 10 minute preincu (iii) bation of the biological sample with a hydrazine prior to lysis. S 0056 FIG. 7 compares adipic acid dihydrazide, succinic - C-: acid dihydrazide, and aminoguanidine with 10 minute and overnight treatments. and 0057 FIG. 8 demonstrates the effect of pretreatment with 0043 m is an integer selected from the group consisting a hydrazine in the presence or absence of proteinase K. of 0 and 1; and 0.058 FIG. 9 demonstrates the performance of succinic 0044 n is an integer selected from the group consisting acid dihydrazide and adipic acid dihydrazide in a QiaAmp of 1 and 2. extraction, where the elution step is performed at either room 0045. In another embodiment, semicarbazide; thiosemi temperature (25°C.) or at 56°C. carbazide; carbazide; thiocarbazide, N-aminoguanidine and a salt thereof, including hydrochloride salts: N,N-diami DETAILED DESCRIPTION noguanidine and a salt thereof, including dihydrochloride salts; acetylhydrazide; adipic acid dihydrazide. Succinic acid 0059. The present application relates generally to the use dihydrazide; formic hydrazide; maleic acid dihydrazide; mal of Scavenger compounds to increase the recovery a target onic acid dihydrazide; benzenesulfonylhydrazide; tosylhy molecule from a biological sample preserved in a liquid cytol drazide; methylsulfonylhydrazide. ogy medium. 0046. In another embodiment, the Scavenging agent of the 0060. In an embodiment, a method is provided for recov lysis solution is selected from the group consisting of adipic ering a nucleic acid or a protein from a biological sample in a acid dihydrazide, Succinic acid dihydrazide, and aminoguani liquid cytological preservative medium, comprising: a) add dine hydrochloride. ing a scavenger agent to the medium; b) incubating at a temperature and for a time sufficient for the Scavenger agent 0047. In another embodiment, the lysis solution comprises to Scavenge a preservative agent in the sample; and c) recov from about 0.2M to about 1.5M of the scavenging agent. ering the nucleic acid or protein. As used herein, a scavenging 0048. In another embodiment, the lysis solution comprises agent is any compound that is capable of reacting with a from about 0.2M to about 0.5M adipic acid dihydrazide or fixative agent, thereby removing the fixative agent from the from about 0.2M to about 0.5M succinic acid dihydrazide. solution. In the context of the presently disclosed materials 0049. In another embodiment, a kit for recovering a target and methods, these compounds ideally exhibit one or more of molecule from a biological sample preserved in a liquid cytol the following properties: (1) highly soluble in water; (2) ogy preservative solution is provided, said kit comprising a chemically neutral; (3) non-toxic; and (4) readily available lysis solution as set forth above and optionally comprising at and inexpensive. In another embodiment, these compounds least one additional component selected from the group con exhibit each of the above properties. sisting of protein digestive enzyme, a solid phase, a nucleic 0061 The liquid cytological preservative medium may acid probe capable of hybridizing to the target nucleic acid, include cross-linking and/or precipitating preservative and an antibody. agents. Cross-linking preservative agents include without limitation aldehydes, osmium tetroxide, potassium dichro BRIEF DESCRIPTION OF THE FIGURES mate, chromic acid, and potassium permanganate. Precipitat 0050 FIG. 1 outlines the basic mechanism by which ing preservative agents include without limitation alcohols hydrazines function as carbonyl scavengers, using ami and acetic acid. noguanidine as an exemplary hydrazine compound. 0062. In one embodiment, the liquid cytological preserva 0051 FIG. 2 illustrates the recovery of a nucleic acid from tive media comprises a carbonyl-based preservative agent, SiHa cells spiked in SurePath liquid cytological collection Such as formaldehyde. Formaldehyde is a cross-reactive mol medium in the presence or absence of three different hydra ecule that fixes cells by cross-linking amino groups by a Zines: adipic acid dihydrazide, Succinic acid dihydrazide, and methylene bridges. Although this is useful for cytology pur amino guanidine. In each instance, the right bar represents the poses, it can inhibit the efficient isolation of nucleic acids result of adding the indicated hydrazine to the indicated final and/or proteins from the fixed samples. US 2011/01961.46 A1 Aug. 11, 2011

0063. In an embodiment, the liquid cytology preservative wherein: medium comprises a aldehyde. Exemplary aldehydes com 0067) R' C-C alkyl: C-C alkenyl: C-C, monly used in liquid cytology preservative medium includes, cycloalkyl, C-C cycloalkenyl; Co-Co aryl; and Co-Co but is not limited to, formaldehyde, glyoxal, glutaraldehyde, heteroaryl; and glyceraldehyde, acrolein, or other aliphatic aldehydes; or 0068 m is an integer selected from the group consisting aldehyde(s) of unknown nature. One such liquid cytological of 0 and 1. preservative medium is SUREPATHR, one of the most com 0069. In one exemplary embodiment, the scavenging monly used preservative media in clinical settings. SURE agent is aminoguanidine, which is a hydrazine according to PATH medium has a nearly 37% formaldehyde content and formula I wherein m is 0. also contains methanol, ethanol, and isopropanol. The high 0070. In other exemplary embodiments, the scavenging formaldehyde content makes it a useful fixative, but poses agent is a hydrazine according to formula I wherein m is 1 and challenges for extracting target molecules (such as nucleic R" is selected from the group consisting of C-C2 alkyl, acids and proteins) from the fixed samples and using them for C-C alkyl, and C-C alkyl. Subsequent molecular analysis. Also included in this embodi 0071. In another embodiment, the scavenging agent is ment are preservative media known to contain formaldehyde selected from the class of hydrazines known as hydrazides. releasers, including but not limited to, Quaternium-15 (meth Hydrazides similarly are capable of reacting with carbonyls enamine-3-chloroallylochloride), Tris Nitro (tris-hydroxym (aldehydes and ketones) to form hydrazone bonds. As used ethylnitromethane), Glydant (1,3-dimethylol-5,5-dimethyl herein, a hydrazide is a compound comprising a terminal hydantoin, or DMDM-hydantoin), Germal-115 (imidazolidi hydrazide functional group: nyl urea), Germall II (diazolidinyl urea), Bronopol (2-bromo 2-nitropropane-1,3-diol), Bronidox (5-bromo-5-nitro-1,3-di oxane), Bromothalonil (methyldibromo glutaronitrile, 1.2- dibromo-2,4-dicyanobutane), Suttocide A (hydroxymethylglycinate), and paraformaldehyde. Also wherein the wavy bond indicates an attachment to the core included in this embodiment are media containing aldehydes chemical structure and R is selected from the group consist of unknown origins, which can be detected by use of alde ing of hyde-specific reagents, such as Purpald (4-Amino-3-hy drazino-5-mercapto-1,2,4-triazole). 0064. In one embodiment, the scavenging agent is capable of reacting with a carbonyl compound, such as an aldehyde. Exemplary carbonyl scavenging agents include, without limi tation, hydrazines. As used herein, a hydrazine is any com b) pound comprising a terminal hydrazine functional group. As used herein a “hydrazine functional group' is a chemical group according to the following formula:

H MAN-NH2, wherein the wavy bond indicates an attachment to the remain der if the chemical structure. Hydrazines readily react with 0072. In one embodiment, the Scavenging agent is a carbonyls (aldehydes and ketones) to form hydraZone bonds. hydrazide according to formula II: These bonds replace the oxygen of the carbonyl with a nitrog enous functional group limiting the ability of the aldehyde to bind with other factors. An exemplary reaction scheme is set (II) forth in FIG. 1. R (R2 N NH2 ) as 0065 Hydrazines may be a unifunctional or multifunc tional. A “unifunctional hydrazine is a hydrazine containing wherein: a single terminal hydrazine functional group. A "multifunc 0073) R' C-C alkyl: C-C alkenyl: C-C, tional hydrazine' is a hydrazine containing more than one cycloalkyl, C-C cycloalkenyl; Co-Co aryl; and Co-Co terminal hydrazine functional group. heteroaryl; wherein R' may optionally be optionally 0.066. In one embodiment, the Scavenging agent is a hydra substituted so as to increase the solubility of the scav Zine according to formula I: enging agent in Water 0074) R' which may be in each instance the same or different and is selected from the group consisting of (I) NH2 (R1 "ss NH2 H NH US 2011/01961.46 A1 Aug. 11, 2011

Solution. In another exemplary embodiment, the Scavenger -continued Solution may be added directly to the liquid cytology preser b) Vative medium. In another exemplary embodiment, the O amount of scavenger Solution to be added may be based on an -S- and estimation of the quantity of fixative agent in the sample. The amount of scavenging agent can be the same as the amount of O fixative agent in the sample, less than the amount of the c) S fixative agent in the sample, or in excess of the fixative agent in the sample. In one embodiment, the amount of scavenging - C-: agent is in slight excess over the amount of fixative agent in the sample. In an exemplary embodiment, the scavenger solu and tion comprises from about 0.1M to about 1.OM, from about 0075 m is an integer selected from the group consisting 0.1M to about 0.5M, from about 0.2M to about 0.4M, or about of 1 and 2. 0.3M of the Scavenging agent. In another embodiment, about 0076. In one exemplary embodiment, the scavenging 0.3M adipic acid dihydrazide or succinic acid dihydrazide is agent is a hydrazide according to formula II wherein R' is used. As used herein, the term “about, when used in the selected from the group consisting of C-C alkyl, C-C, context of a concentration, expressly includes all concentra alkyl, and C-C alkyl. tions which can be rounded up or down to the indicated 0077. In another exemplary embodiment, the scavenging concentration. agent is a difunctional hydrazide according to formula II I0083. In another embodiment, the target molecule may be wherein n is 2 and R' is selected from the group consisting of extracted from the biological sample by a method compris C-C alkyl, C-C alkyl, and C-C alkyl. ing, inter alia, lysis. As used herein, the terms “lysis' and 0078. In another embodiment, the scavenging agent is a “lysing” refer to the act of disrupting the integrity of a cell hydrazine is selected from carbonic and thiocarbonic acid wall; a cell membrane; or an organelle or other structure derivatives, including but not limited to: semicarbazide defined by a lipid membrane, including but not limited to an (chemical formula: NHC(=O)NHNH); thiosemicarbazide endoplasmic reticulum, Golgi apparatus, lysosome, mito (chemical formula: NHC(=S)NHNH); carbazide (chemi chondrion, nucleus, vacuole, and vesicle. Exemplary meth cal formula: NH-NHC(=O)NHNH); thiocarbazide (chemi ods of lysis include mechanical lysis, such as by Sonication or cal formula: NH-NHC(=O)NHNH); N-aminoguanidine cytolysis; and chemical lysis, including use of detergents and a salt thereof, including hydrochloride salts, and N.N- Such as polyoxyethyleneglycol dodecyl ether (sold commer diaminoguanidine (chemical formula: NHNHC(=NH) cially as Brij-58), 3-(3-cholamidopropyl)dimethylammo NHNH), and a salt thereof, including dihydrochloride salts. nio-1-propanesulfonate (sold commercially as CHAPS), 0079. In other exemplary embodiments, the scavenger Nonidet P-40 (also known as Igepal CA-630), deoxycholate, agent is a hydrazine selected from the group consisting of Triton X-100, sodium dodecyl sulfate (sold commercially as acetylhydrazide (chemical formula: CHC(=O)NHNH), SDS), and/or polysorbate surfactants (sold commercially as adipic acid dihydrazide (chemical formula: NHNHC(=O) TWEEN). (CH2)C(=O)NHNH), succinic acid dihydrazide, (chemi I0084. When the target molecule is extract by lysis, the cal formula: HNNHCOCH-CHCONHNH), formic biological sample should be contacted with a lysis solution. hydrazide (chemical formula: NH-NHC(=O)H), maleic As used herein, “lysis solution” refers to any solution that is acid dihydrazide (chemical formula: useful for lysing a cell. Exemplary lysis Solutions include HNNHCOCHCHCONHNH), and malonic acid dihy without limitation hypotonic lysis solutions and detergent drazide (chemical formula: HNNHCOCHCONHNH). based lysis solutions, including but not limited to lysis solu 0080. In other exemplary embodiments, the scavenger tions comprising polyoxyethylene (20) cetyl ether (sold com agent is a hydrazine selected from Sulfonic acids derivatives, mercially aS Brij-58), 3-(3-cholamidopropyl) including but not limited to, benzenesulfonylhydrazide dimethylammonio-1-propanesulfonate (sold commercially (chemical formula: HNNHS(=O)CH), tosylhydrazide as CHAPS), Nonidet P-40 (also known as Igepal CA-630, (chemical formula: HNNHS(=O)CHCH), and methyl tert-octylphenoxy poly(Oxyethylene)ethanol), deoxycholate, sulfonylhydrazide (chemical formula: HNNHS(=O) Triton X-100, sodium dodecyl sulfate (sold commercially as 2CH3). SDS), and/or polysorbate surfactants (sold commercially as 0081. In certain embodiments, the scavenging agent may TWEEN). be modified by methods known in the art to increase the I0085. In another embodiment, the lysis solution may solubility of the scavenging agent in water. For example, R' in optionally comprise a buffering agent. Exemplary buffering according to formula I or formula II optionally may be Sub agents include without limitation tris(hydroxymethyl)ami stituted with constituents that increase the hydrophilicity of nomethane (“TRIS) and derivatives thereof, such as N-tris the R' constituent. (hydroxymethyl)methyl-3-aminopropanesulfonic acid 0082 In one embodiment, the method comprises contact (“TAPS), 3-N-tris-(hydroxymethyl)-methyl-amino-2-hy ing the biological sample with a scavenger Solution. As used droxypropanesulfonic acid (“TAPSO); N-tris(hydroxym herein, the term "scavenger Solution” refers to an aqueous ethyl)methyl-2-aminoethanesulfonic acid (“TES); N-tris Solution comprising the scavenging agent. In an exemplary (hydroxymethyl)methyl-glycine (“TRICINE); bis(2- embodiment, the biological sample may be separated from hydroxyethyl)iminotris-(hydroxymethyl)methane (“bis the liquid cytology preservative medium and then contacted TRIS”); 1,3-bis(tris(hydroxy-methyl)methylaminopropane with the scavenger solution. In Such a case, the biological (“bis-TRIS PROPANE'); carbonate-bicarbonate; glycine; sample may be incubated in a single aliquot of the scavenger phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic Solution or may be washed sequentially in the scavenger acid (“HEPES); N,N-bis(2-hydroxyethyl)glycine (“Bi US 2011/01961.46 A1 Aug. 11, 2011 cine); 3-(N-morpholino)propanesulfonic acid (“MOPS): tubes; magnetizable or magnetic (e.g. paramagnetic, Super and other Good buffers. Numerous other constituents of the paramagnetic, ferromagnetic or ferrimagnetic) particles, lysis solution may be included as well. The precise type and including but not limited to polystyrene, agarose, polyacry formulation of the lysis solution can be readily determined by lamide, dextran, and/or silica materials having a magnetic a person having ordinary skill in the art according to the material incorporated therein or associated therewith. In sample type, the method of lysis, the analyte of interest, and Some exemplary embodiments, the nucleic acid probe oranti body can be linked to the Surface of a processing vessel Such the method of analysis to be used. as a micro-tube, a well of micro-plate, or capillary, and using I0086. In an embodiment, the scavenger solution may also these Surfaces the nucleic acid can be isolated on a micro function as the lysis solution. Alternatively, the scavenger scale. Where a biotinylated nucleic acid probe or antibody is Solution and the lysis solution may be added to the biological provided, the Solid phase may be coated with a Substance sample as separate solutions, either sequentially or at the capable of binding the biotin moiety, Such as, for example, same time. In one embodiment, the biological sample is incu avidin, Streptavidin, and/or neutravidin. In another embodi bated with the scavenger solution before the lysis solution is ment, the Solid phase may be coated with, or adapted to be added. In another embodiment, the scavenger Solution is coated with, an antibody specific for a DNA:RNA hybrid. removed before the lysis solution is added. In another 0090 Nucleic acids obtained using the disclosed methods embodiment, the lysis solution is added to the biological and compositions may be used in Subsequent molecular ana sample before the Scavenger solution is added. In another lytical methods including without limitation gel electro embodiment, the biological sample may be incubated with phoresis, PCR-related techniques including reverse tran the lysis solution under conditions sufficient to either partially Scriptase PCR and real time PCR, sequencing, Sub-cloning or completely release the target molecule from the biological procedures, Southern blotting, northern blotting, fluorescent sample before the scavenger solution is added. in situ hybridization, and various mutational analyses includ 0087. In one embodiment, the target molecule is a nucleic ing hybrid capture and multiplex analysis. acid. Any method may be used to recover the nucleic acid from the biological sample. By way of example, methods of 0091. In one embodiment, the scavenger solution com recovering the target nucleic acid include without limitation: prises all components necessary for releasing the target chromatography, including but not limited to silica or glass nucleic acid from the biological sample and isolating the adsorption, ion exchange chromatography, affinity purifica target molecule. By way of example and not limitation, Such a scavenger solution could comprise, interalia, a detergent, a tion, spin column chromatography, and gel filtration; Solvent buffer, a nucleic acid probe specific for the target nucleic acid, extraction and precipitation; and centrifugation. Nucleic acid and a solid phase, wherein hybridization of the nucleic acid to recovery methods include without limitation ammonium sul the nucleic acid probe captures the target nucleic acid to the fate precipitation, differential solubilization, Sucrose gradient Solid phase. By way of example and not limitation, this could centrifugation, and chromatography. By way of example and be achieved by modifying the nucleic acid probe to comprise not limitation, the nucleic acid may be isolated by using aligand capable of binding to a constituent of the Solid phase. magnetic beads modified to bind specifically to nucleic acids. For example, the probe may be modified to contain a biotin 0088. In another embodiment, a nucleic acid comprising a moiety and the Solid phase may be coated with a Substance specific sequence may be isolated by hybridizing it to a capable of binding the biotin moiety, Such as, for example, nucleic acid probe complementary to the specific sequence. avidin, Streptavidin, and/or neutravidin, or a fragment In one embodiment, the nucleic acid probe is bound to a solid thereof. In another example, hybridization of the target phase or adapted to be bound to a solid phase. In another nucleic acid to the nucleic acid probe may form a DNA:RNA embodiment, hybridization of the nucleic acid probe to the hybrid, which then may be captured to the solid phase via nucleic acid molecule results in a DNA:RNA hybrid between the probe and the nucleic acid molecule. The resulting hybrid antibodies specific for DNA:RNA hybrids, such as by the may then be bound by an antibodies known to bind specifi methods described in U.S. Pat. No. 6.228,578 and U.S. patent cally to DNA:RNA hybrids (“DNA:RNA-binding anti application Ser. No. 12/695,071, the contents of which are body'), which in turn may be bound to a solid phase or incorporated in their entirety by reference. adapted to be bound to a solid phase. In either case, hybrid 0092. In one embodiment, the target molecule is a ization of the probe with the nucleic acid results in the nucleic polypeptide. As used herein, the term “polypeptide' refers to acid being associated with a solid phase, which may then be any molecule comprising at least two amino acids joined by a separated from the lysate using mechanical means. By way of peptide bond, and expressly includes oligopeptides and pro example and not limitation, Such methods are described in teins. Polypeptide recovery methods include without limita U.S. Pat. No. 6.228,578 and U.S. patent application Ser. No. tion ammonium Sulfate precipitation, differential solubiliza 12/695,071, the contents of which are incorporated in their tion, Sucrose gradient centrifugation, and chromatography. entirety by reference. Exemplary DNA:RNA-binding anti Chromatographic polypeptide isolation methods include bodies include, but are not limited to, those disclosed in U.S. without limitation size exclusion, ion exchange, hydrophobic Pat. Nos. 4,732,847 and 4,865,980, the contents of which are interaction, affinity, immuno-affinity, and metal binding incorporated herein by reference in their entireties. chromatography. 0089. By way of example, and not limitation, an appropri 0093 Polypeptides obtained with the disclosed methods ate solid phase includes, but is not limited to: silica, borosili and compositions may be used in Subsequent molecular ana cates, silicates, anorganic glasses, organic polymers such as lytical methods including without limitation sequencing, poly(meth)acrylates, polyurethanes, polystyrene, agarose, immunoprecipitation, western blots, ELISA assays, dot blots, polysaccharides Such as cellulose, metal oxides Such as alu and enzyme assay minum oxide, magnesium oxide, titanium oxide and Zirco 0094. The methods described also can be used to isolate nium oxide, metals such as gold or platinum, agarose, Sepha whole pathogens, including without limitation bacteria, dex, Sepharose, polyacrylamide, divinylbenzene polymers, fungi, yeast, protozoa, prions, and viruses. styrene divinylbenzene polymers, dextrans, and derivatives 0.095 The methods and compositions described herein are thereof, and/or silica gels, beads, membranes, and resins; easily and rapidly optimized for specimens preserved in glass or silica Surfaces, such as beads, plates, and capillary either cross-linking or precipitating fixatives. The methods US 2011/01961.46 A1 Aug. 11, 2011

and compositions described herein also are adaptable for all (0103 C. General Protocol: QiaAmp(R) Extraction biological fluids and provide simple protocols that are proven 0104. In some examples, nucleic acid extraction was per compatible with high throughput automation, including for formed using a QiaAmpR DNA extraction kit (Qiagen Inc., example the QIAensemble(R) NextGenTM Sample Processor, Hilden, Germany), which is a commercially available DNA an automated sample processing device for extraction and extraction technology based on silica gel absorption in a analysis which provides full automation, including de-cap column format. ping and capping of specimens and Zero ergonomic move 0105 250 ul of a SurePath R sample and 50 uL of an ments. As such, they provide ultra high through-put and eco aqueous solution of the scavenging agent (or ultrapure water) logically friendly sample processing by allowing for a were added to a 2 mL tube and briefly vortexed to mix. 80 LL flexible input Volume, non-hazardous material liquid waste, of buffer ATL (provided with the kit) and 20 uL of a protein limited Solid waste, and reagents that may be stored at room ase K solution (provided with the kit) were added to the tube, temperature. the tube was pulse vortexed 10 times, and then incubated at 70° C. in a shaking water bath (900 rpm) for 15 minutes. 360 EXAMPLES uL of 100% ethanol was added, the tube was pulse vortexed 15 times, and the mixture was incubated at room temperature Materials and Methods for 5 minutes. The lysate was then transferred to a Qia Amp(R) 0096 A. Cells and Samples column placed on a QiaVacR vacuum manifold. The lysate 0097 Biological samples comprising human papillomavi was then pulled through the column by application of rus 16 nucleic acids were used to test the materials and meth vacuum. The column was then washed twice, first with 750 ods according to the present disclosure. In some examples, a uL of buffer AW2 (provided with the kit) and then with 100% SiHa cell line is used, which are derived from a human cer ethanol. Columns were then removed from the manifold, Vical carcinoma. SiHa cells are known to contain an inte placed in 2 mL tubes, and centrifuged at 14000 rpm for 3 grated HPV 16 genome. In other examples, clinical samples minutes. The columns are then placed in elution tubes and determined to be infected with HPV 16 were used. As nega dried at 56°C. for 3 minutes. 70 uL of buffer AVE were then tive controls for the HPV 16 assay, Jurkat cells or clinical added to the membrane, incubated at room temperature for 5 samples determined to be HPV negative were used. minutes, and centrifuged at 14000 rpm for 1 minute. The 0098. Unless otherwise indicated, all samples used in the eluate was transferred to a 96 well plate and the presence of present examples were fixed at least overnight in the indicated HPV 16 was detected using either the NexGen(R) (described liquid cytology preservative medium. in, inter alia, U.S. Patent Application Publication No. 2010/ 0099 B. General Protocol: Handy LabsTM Extraction 0105060A1) or HC2(R) assay (described in U.S. Pat. No. 0100. In some examples, extraction was performed using 6.228,578). the Handy LabsTM extraction technology, which is a commer Example 1 cially available DNA extraction technology based on anion 0106. In this example, the initial feasibility of using hydra exchange chromatography, using paramagnetic beads treated zine compounds is carried out using SUREPATH clinical with an anion exchange material. negatives spiked with 10,000 SiHa cells (HPV 16+). Samples 0101 The following general protocol was used in the comprising only the clinical negatives are used as negative present examples. controls for each condition. Each sample was treated with 0102 Samples were spiked in 500 uL SurePath(R) liquid adipic acid, lysed, and processed for nucleic acid isolation cytological medium and placed in 5 mL tubes. 100LL of an using the Handy Labs protocol set forth above. Unless other aqueous solution of the scavenging agent (or ultrapure water) wise indicated, each sample contains proteinase K. and a proteinase K tablet (provided with the kit) were added 0107 As set forth in Table I, “CV refers to the coefficient to the tube. 1.2 mL of lysis buffer and 60LL of HL beads (both of variation, “S/N refers to the signal to noise ratio, “RLU/ provided with the kit) were then added and tube was incu CO” refers to the Relative light units/cutoff ratio, and “PK” bated at 60° C. for 10 minutes. A magnet was then added for refers to proteinase K. 1 pg of a plasmid comprising the entire 2 minutes to pellet the beads and the Supernatant removed. HPV 16 genome was used as a positive control for the Nex The beads were then resuspended in 400 uL of buffer 1 Gen(R) assay. The baseline for percent recovery is established (supplied with the kit), the mixture shaken, the beads pelleted, using SiHa cells spiked in Jurkat cells without a liquid cytol and the Supernatant removed. Then, the beads were resus ogy preservative medium. “% Recovery A' represents data pended in 30 uL of buffer 2 (supplied with the kit) and the obtained using 0.3Madipic acid dihydrazide without protein nucleic acid eluted 65.8° C. for 9 minutes. Beads were pel ase K compared to the baseline. “% Recovery B represents leted again and the eluate transferred to a 96 well plate. 12 uL data obtained using 0.3M adipic acid dihydrazide with pro of buffer 3 (supplied with the kit) and 8 uI of Digene Collec teinase K compared to the baseline. “96 Recovery C repre tion Medium (Qiagen Gaithersburg, Inc., Qiagen, MD) were sents data obtained using 0.16Madipic acid dihydrazide with added to each well. The presence of HPV 16 was detected proteinase K compared to the baseline. “96 Recovery D' using either the NexGen(R) (described in, inter alia, U.S.) or represents data obtained using 0.3M adipic acid dihydrazide HC2(R) assay (described in U.S. Pat. No. 6.228,578). without proteinase K compared to the baseline.

TABLE 1 Target Probe B + 15 ng Avg. CV SiN S-N

Negative 53 55 53 59 55 59 Calibration US 2011/01961.46 A1 Aug. 11, 2011

TABLE 1-continued

Detection 39 41 41 45 42 6% O.8 -14 Reagent 1 HPV 16-1pg 275 263 301 289 282 6% S.1 227

Direct Controls Avg. CV SiN S-N RLUCO

Jurkat 63 59 55 55 58 79% 1.O O O.21 2OK 1453 5151 2O67 941 2403 79% 414 2345 8.52 SiHa 500 ul 0.3M Adipic Acid 0.05M (Final) without PK Avg CV S/N S-N RLUCO Negative 145 51 87 94 SO% 1.O O O.33 Clinical 2OK 117 121 121 120 290 1.3 25 0.4 SiHa 500 ul 0.3M Adipic Acid 0.05M (Final) with PK Avg CV S/N S-N RLUCO Negative 63 77 93 78 1990 1.O O O.28 Clinical 2OK 2525 2845 2S79 26SO 6%. 34.1 2572 9.4 SiHa 500 ul 0.16M Adipic Acid 0.027M (Final) with PK Avg CV S/N S-N RLU/CO Negative 57 79 67 68 16% 1.O O O.24 Clinical 2OK 2S11 2655 2447 2S38 4%. 37.5 2470 9.0 SiHa 500 ul 0.OM Adipic Acid O.OM (Final) with PK Avg CV S/N S-N RLUCO

Negative 53 65 69 62. 13% 1.O O O.22 Clinical 2OK 1673 2081 1997 1917 1190 30.8 1855 6.8 SiHa 500 ul % Recovery A % Recovery B % Recovery C % Recovery D

196 1.10% 105% 79%

Example 2 0108. When amino guanidine hydrochloride is used as a scavenger agent in SUREPATH media with pre-incubation period of 10 minutes, 95% to 100% recovery of DNA is obtained.

TABLE 2

500 ul of water or SP Water water ISP SP SP SP SP SP

LB (1:2) ul 2000 2OOO 1200 1200 1400 1600 PK (20 mg/ml)u is 20 0 12 14 16 18 20 Negative Spiked 25000 SiHa cells spiked into 0.5 ml of negative SP pool. Cells fixed for Pool SurePath 10 days. Final cell vol. was 0.5 ml of SP or water. Each tube extracted SurePath using anion exchange bead method. 5K cells used per well. 3 4 5 6 7 8 9 10 11 12 US 2011/01961.46 A1 Aug. 11, 2011

TABLE 2-continued

DCM-1PG Calibrator Calibrator

69 103 99 113 95 105 91 315 87 123 99 10 913 883 1239: 27 1 89 9 23 887 989 Avg (A-D) 88.33 O5.7 100 O1.5 8O 101.7 94.3 99 87 CV (A-D) 11.2 8.5 6.2 16.9 15.9 6 6.8 13.2 11.3 395.5 305.5 CV (E-H) 11.8 12.6 8.6 21.1 SN 3.1 12.8 11.1 7.1 % Recovery to DCM/SiHA 110.8% 105.0% 112.4% 62.4% Shaded/Col. 2, Avg. (E-H) % Recovery to 1PG 249.6% 236.5% 253.2% 140.6% Shaded/Col. 1, Avg. (E-H) % Recovery to DCM/SiHa 335.0% 317.3% 339.8% 188.7% Shaded/Col. 4, Avg. (E-H) % Recovery to 1 PG 42.0% 258.8% 263.6%. 229.2% 223.1% 245.1% 262.5% 145.8% Shaded/Col. 3, Avg. (E-H) % 42.83 ver 110.83 112.89 98.14 95.56 104.98 recovery-2 0% 38.1 29.43 5.99 98.6 100.43 87.32 85.02 93.4 recovery 12

0109 When amino guanidine hydrochloride is added, the Handy Labs protocol. 20,000 SiHa cells were spiked in either solution can be thoroughly mixed. The SUREPATH speci SurePath medium or Digene Collection Medium and pro men can be at room temperature before mixing with amino cessed according to the Handy Labs protocol set forth above. guanidine hydrochloride. Approximately 300 uL to 400 uL of Duplicate experiments were performed for each condition. amino guanidine hydrochloride (0.5M) can be added to about Results for each hydrazine in SurePath medium are illustrated 500LL of SUREPATH media used in assays described herein. at FIG. 2. As can be seen, in each case the addition of Scav If it is beneficial to reduce working Volume, amino guanidine enger improved recovery of the nucleic acid. When compared hydrochloride at higher concentration can be used. Since to a sample in Digene Collection Medium, percent recovery amino guanidine hydrochloride is highly soluble a concen in the absence of scavenger varied between 40% and 60%, trated (e.g., 4M) solution can be prepared. In an embodiment, while addition of the scavenger improved recovery to a stock scavenger solution comprising 1M, 2M, 3M, or 4M between 85% and 100%. amino guanidine hydrochloride can be used. 0110. As set forth in Table 2, “CV refers to the coefficient of variation, “NC refers to negative calibration, “PK” refers Example 4 to proteinase K, “DCM” refers to the DIGENE collection medium, “DCM-1PG' refers to the DIGENE collection 0112 Various concentrations of succinic acid dihydrazide medium with 1 pg of positive calibrator, “SP” refers to SURE and adipic acid dihydrazide were also tested in the Handy PATH media, “NP-SP refers to Negative Pool SUREPATH Labs protocol. media, and “Sp-SP” refers to Spiked SUREPATH media. 0113. In one experiment, 20,000 SiHa cells were spiked into SurePath medium and processed according to the Handy Example 3 Labs protocol set forth above, using 0.16M, 0.3M, or 0.5M 0111 Aminoguanidine, adipic acid dihydrazide, and Suc Succinic acid dihydrazide or an equal Volume of water. cinic dihydrazide were compared to one another in the Results are demonstrated at FIG. 3. US 2011/01961.46 A1 Aug. 11, 2011

0114. In another experiment, 20,000 SiHa cells were a) a compound according to formula I: spiked into SurePath medium and processed according to the Handy Labs protocol set forth above, using 0.16M, 0.3M, or (I) 0.5M adipic acid dihydrazide or an equal volume of water. NH2 (R'). NH2: Results are demonstrated at FIG. 4. H NH Example 5 and 0115 The effect of varying the cell number (and therefore b) a compound according to formula II: the copy number of the target nucleic acid) was also tested. Stocks of 20,000, 10,000, 5,000, 2,500, and 0 SiHa cells per 500 uL of SurePath were generated and processed according (II) to the Handy Labs protocol using either 0.3M adipic acid R!If (R4-N-NH), D2- H dihydrazide or an equal volume of water. Results are shown at wherein: FIG.S. R" C1-C alkyl: C-C2 alkenyl: C-C cycloalkyl; C-C cycloalkenyl: Co-Co aryl; and Co-Co het Example 6 eroaryl; 0116. Whether pretreatment with a scavenging agent R’ which in each instance may be the same or different, improves recovery was also tested. 20,000 SiHa cells were and is selected from the group consisting of: spiked into either SurePath medium or Digene Collection Medium and processed according to the Handy Labs protocol (i) set forth above, except that some samples were incubated with either 0.3M adipic acid dihydrazide or 0.5M ami noguanidine (or an equal Volume of water) for 10 minutes (ii) before addition of the proteinase K and lysis buffer. This was repeated using either 10 minute or overnight preincubation with 0.3M adipic acid dihydrazide, 0.3M Succinic acid dihy drazide, or 0.5M aminoguanidine. Results are demonstrated at FIG. 6 and FIG. 7. (iii) 0117. Additionally, it was tested whether inclusion of pro teinase K in the pretreatment would have an effect. Samples were tested as above using 0.3M succinic acid dihydrazideas the Scavenger, except one sample was pretreated for 10 min m is an integer selected from the group consisting of 0 utes with a scavenger Solution comprising proteinase K. and 1; and Results are shown at FIG. 8. n is an integer selected from the group consisting of 1 and 2: Example 7 wherein the Scavenging agent is optionally modified so as 0118. It has been observed that the QiaAmp protocol is to increase solubility in water. less compatible with SurePath medium than the Handy Labs 3. The method of claim 2 wherein the Scavenging agent is protocol. It was therefore investigated whether increasing the a compound of formula I wherein R' is C-C alkyland mis temperature at which the nucleic acid is eluted from the anion 1. exchange column could improve recovery. 20,000 SiHa cells 4. The method of claim3 wherein R' is C-C alkyl. were spiked into 500 uL of SurePath medium and processed 5. The method of claim 4 wherein R' is C-C alkyl. according to either the HandyLabs protocol or the QiaAmp 6. The method of claim 2 wherein the Scavenging agent is protocol set forth above using either 0.3M Succinic acid dihy a compound of formula II wherein R' is C-C alkyl; R is drazide or 0.3M adipic acid dihydrazide was the scavenger. O For the QiaAmp protocol, elution was performed at either room temperature or 56°C. Results are shown at FIG. 9. -C-: and n is 2. What is claimed is: 7. The method of claim 6 wherein R' is C-C alkyl. 1. A method for extracting a target molecule from a bio 8. The method of claim 6 wherein R' is C-C alkyl. logical sample preserved in a liquid cytology preservative 9. The method of claim 1 wherein the Scavenging agent is Solution, the method comprising: selected from the group consisting of semicarbazide; thi A) contacting the biological sample with a scavenger Solu osemicarbazide; carbazide; thiocarbazide, N-aminoguani tion comprising a scavenging agent comprising at least dine and a salt thereof. N.N-diaminoguanidine and a salt one terminal hydrazine group; thereof acetylhydrazide, adipic acid dihydrazide. Succinic B) treating the biological sample under conditions suffi acid dihydrazide; formic hydrazide; maleic acid dihydrazide: cient to release the nucleic acid or protein from the malonic acid dihydrazide; benzenesulfonylhydrazide; tosyl biological sample; and hydrazide; methylsulfonylhydrazide. C) isolating the target molecule. 10. The method of claim 1 wherein the scavenger solution 2. The method of claim 1, wherein the scavenging agent is comprises from about 0.1M to about 1.OM of the scavenging selected from the group consisting of: agent. US 2011/01961.46 A1 Aug. 11, 2011

11. The method of claim 10 wherein the scavenger solution a) a compound according to formula I: comprises from about 0.1M to about 0.5M of the scavenging agent. 12. The method of claim 10 wherein the scavenger solution (I) comprises from about 0.2M to about 0.4M of the scavenging NH2 agent. 13. The method of claim 1 wherein the scavenger solution NH comprises about 0.3Madipic acid dihydrazide or about 0.3 M Succinic acid dihydrazide. and 14. The method of claim 1 wherein the scavenger solution b) a compound according to formula II: is added directly to the liquid cytology preservative solution. 15. The method of claim 1 wherein the scavenger solution further comprises a protein digestive enzyme. (II) 16. The method of claim 1 wherein the biological sample is R (R2 N NH ) contacted with the Scavenger Solution before the target mol 2 as ecule is released from the biological sample. wherein: 17. The method of claim 1 wherein the target molecule is R" is selected from the group consisting of C-C alkyl; released from the biological sample by lysing the biological C-C alkenyl: C-C cycloalkyl, C-C cycloalkenyl: sample in the presence of a lysis solution. Co-Co aryl; and Co-Co heteroaryl, wherein R' is 18. The method of claim 17 wherein the scavenger solution optionally substituted so as to increase the solubility of is the lysis solution. the Scavenging agent in water; 19. The method of claim 17 wherein the scavenger solution is added to biological sample before, after, or at the same time R’ which in eachinstance may be the same or different, and as the lysis solution is added to the biological sample. is selected from the group consisting of 20. The method of claim 1 wherein the target molecule is a (i) target nucleic acid and: O C) the target nucleic acid is isolated by a method compris - C-: 1ng: (ii) (i) hybridizing a nucleic acid probe to the target nucleic O acid with a second nucleic acid to form a nucleic acid hybrid; -S- and (ii) binding the nucleic acid hybrid to a solid phase; O (iii) (iii) isolating the Solid phase; and S (iv) eluting the target nucleic acid from the Solid phase. 21. The method of claim 20 wherein the nucleic acid hybrid - C - and is a DNA:RNA hybrid and wherein the DNA:RNA hybrid is m is an integer selected from the group consisting of 0 and bound to the Solid phase by a method comprising contacting 1; and the nucleic acid hybrid with an antibody capable of binding to n is an integer selected from the group consisting of 1 and the nucleic acid hybrid, wherein the antibody is bound to the 2. solid phase or adapted to be bound to the solid phase. wherein the scavenging agent is optionally modified so as to 22. The method of any of claim 1 wherein the target mol increase solubility in water. ecule is a target nucleic acid and: 27. The lysis solution of claim 25 wherein the scavenging C) the target nucleic acid is isolated by a method compris agent is selected from the group consisting of semicarbazide; ing: thiosemicarbazide; carbazide; thiocarbazide, N-aminoguani (i) binding the target nucleic acid to an anion exchange dine and a salt thereof. N.N-diaminoguanidine and a salt matrix; and thereof acetylhydrazide, adipic acid dihydrazide. Succinic (ii) eluting the target nucleic acid from the anion acid dihydrazide; formic hydrazide; maleic acid dihydrazide: exchange matrix. malonic acid dihydrazide; benzenesulfonylhydrazide; tosyl 23. The method of claim 22 wherein the target nucleic acid hydrazide; methylsulfonylhydrazide. is eluted from the anion exchange matrix at an elution tem 28. The lysis solution of claim 25 comprising from about perature of from about 20° C. to about 70° C. 0.1M to about 1.OM of the scavenging agent. 24. The method of claim 22, wherein the target nucleic acid 29. The lysis solution of claim 25 comprising from about is eluted from the anion exchange matrix at an elution tem 0.1M to about 0.5M adipic acid dihydrazide or from about perature of from about 50° C. to about 60° C. 0.1M to about 0.5M succinic acid dihydrazide. 30. A kit for recovering a target nucleic acid from a bio 25. A lysis solution comprising: logical sample preserved in a liquid cytology preservative (i) a buffer; Solution, said kit comprising a lysis solution according to (ii) a detergent; claim 25 and optionally comprising at least one additional (iii) a Scavenging agent comprising at least one terminal component selected from the group consisting of a protein hydrazine group; and digestive enzyme, a Solid phase, a nucleic acid probe capable (iv) optionally, protein digestive enzyme. of hybridizing to the target nucleic acid, and an antibody. 26. The lysis solution of claim 25 wherein the scavenging agent is selected from the group consisting of: c c c c c