US010323241B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 323 ,241 B2 Tsukamoto ( 45 ) Date of Patent : Jun . 18 , 2019 (54 ) METHOD FOR RECOVERING (56 ) References Cited SHORT- CHAIN NUCLEIC ACIDS U . S . PATENT DOCUMENTS (71 ) Applicant: TOPPAN PRINTING CO . , LTD ., 5 ,234 ,809 A 8/ 1993 Boom et al. Tokyo ( JP ) 6 , 180 ,778 B1 1 / 2001 Bastian et al. 9 , 809 ,612 B2 * 11 / 2017 ITILLRitt + + + + + + + + + + + + + ...... CO7H 1 /08 (72 ) Inventor: Kei Tsukamoto , Tokyo (JP ) 2011/ 0130558 A1 6 / 2011 Ritt et al. ( 73 ) Assignee : TOPPAN PRINTING CO . , LTD ., FOREIGN PATENT DOCUMENTS Tokyo ( JP ) 9 - 508638 9 / 1997 JP 2011 - 522529 8 / 2011 ( * ) Notice : Subject to any disclaimer, the term of this WO WO 93 / 11221 A1 6 / 1993 patent is extended or adjusted under 35 WO WO 2007 /065934 A1 6 / 2007 U . S . C . 154 (b ) by 239 days . WO WO 2013 /037401 A1 3 /2013 (21 ) Appl. No. : 15 /292 ,880 OTHER PUBLICATIONS ( 22 ) Filed : Oct . 13 , 2016 International Search Report dated Jul . 21, 2015 in corresponding International Application No . PCT/ JP2015 / 061882 . (65 ) Prior Publication Data US 2017 /0029808 A1 Feb . 2 , 2017 * cited by examiner Related U . S . Application Data Primary Examiner - Lawrence E Crane (63 ) Continuation of application No . (57 ) ABSTRACT PCT/ JP2015 /061882 , filed on Apr. 17 , 2015 . A method of recovering short - chain nucleic acids having a length of 1000 bases or less from a whole blood sample , ( 30 ) Foreign Application Priority Data which includes bringing a first mixture including a whole blood sample into contact with a first nucleic acid adsorbent Apr. 18 , 2014 ( JP ) ...... 2014 -086247 to adsorb nucleic acids derived from the whole blood sample , removing the first nucleic acid adsorbent from being (51 ) Int. Ci. in contact with the first mixture to recover liquid compo CO7H 1 /08 ( 2006 .01 ) nents of the firstmixture , bringing a second mixture includ C12Q 1 /68 ( 2018 .01 ing the recovered liquid components into contact with a C12N 15 / 10 ( 2006 . 01) second nucleic acid adsorbent to adsorb short- chain nucleic C12Q 1/ 6806 ( 2018 . 01 ) acids having a length of 1000 bases or less from the whole (52 ) U . S . CI. blood sample , separating the second nucleic acid adsorbent CPC ...... C12N 15 / 1006 ( 2013 . 01) ; C07H 1/ 08 from being in contact with the second mixture , and eluting ( 2013. 01 ) ; C12Q 1/ 68 (2013 .01 ) ; C12Q the short - chain nucleic acids absorbed in the second nucleic 1 /6806 ( 2013 .01 ) acid adsorbent and recovering the short - chain nucleic acids (58 ) Field of Classification Search from the second nucleic acid adsorbent. CPC ...... C12N 15 /1006 ; C12Q 1/ 68 ; CO7H 1 /08 See application file for complete search history . 12 Claims, 3 Drawing Sheets U .S . Patentatent JumJun. . 18 18, , 20192019 Sheet 1 of 3 US 10 , 323, 241 B2

FIG . 1 M S

10kbp 3kbp 2kbp IN 1 kbp U . S . Patent Jun . 18, 2019 Sheet 2 of 3 US 10 , 323, 241 B2

FIG . 2

1500bp 1000bp 500bp 400bp 300bp 200bp 100bp M S U . S . Patent Jun . 18, 2019 Sheet 3 of 3 US 10 , 323, 241 B2

FIG . 3

3kbp

2kbpma 1kbp M US 10 , 323 , 241 B2

METHOD FOR RECOVERING containing nucleic acids is dissolved if necessary and then SHORT -CHAIN NUCLEIC ACIDS brought into contact with a nucleic acid adsorbent to thereby adsorb nucleic acids in the sample onto the nucleic acid CROSS REFERENCE TO RELATED adsorbent. The nucleic acids adsorbed onto the nucleic acid 5 adsorbent are eluted by a suitable buffer from the nucleic APPLICATIONS acid adsorbent. A typical example of such a method may be a Boom method ( see , for example , Patent Document 1 ( U . S . This application is a continuation application based on a Pat. No . 5 , 234 , 809 ) . ) . The Boom method is a method in PCT Patent Application No. PCT/ JP2015 / 061882 , filed Apr. which a sample containing nucleic acids is incubated with a 17 , 2015 , whose priority is claimed on Japanese Patent chaotropic buffer and a DNA -binding solid phase (nucleic Application No. 2014 -086247 , filed Apr. 18 , 2014 , the 10 acid adsorbent) to thereby isolate nucleic acids from the content of which is hereby incorporated by reference. sample . Both extraction of nucleic acids from cells in the sample and adsorption of the extracted nucleic acids onto a BACKGROUND OF THE INVENTION nucleic acid adsorbent are realized by a chaotropic buffer. As a method similar to the above - described method , for Field of the Invention 15 example , Patent Document 2 (PCT International Publication The present invention relates to a method for efficiently No . WO 1993 / 11221 ) also discloses a method in which a recovering nucleic acids having a relatively short chain sample containing nucleic acids is brought into contact with length such as circulating DNA , from a whole blood sample an anion exchanger in a low ionic strength buffer to adsorb containing a large amount of long - chain nucleic acids such nucleic acids in the sample , the nucleic acids eluted from the as genomic DNA . 20 anion exchanger using a high ionic strength buffer are Description of Related Art adsorbed again onto an inorganic carrier member in the Currently, free nucleic acids circulating in whole blood presence of a lower alcohol, and then the nucleic acids are (peripheral blood ) have attracted much attention in the recovered by water or a low ionic strength buffer from the cancer diagnostic field . Circulating nucleic acids (DNA inorganic carrier member . RNA ) in whole blood are used for various purposes , such as 25 Nucleic acids having a relatively longer chain length than for diagnosing carcinomas and residual lesions associated nucleic acids having a short chain length are readily with carcinomas, for assessing the prognosis of carcinomas, adsorbed onto a nucleic acid adsorbent. Therefore, in the for monitoring the disease course of individuals affected by case where nucleic acids are recovered from a whole blood carcinomas , or for observing the effects of anti- cancer sample using such a method employing a nucleic acid therapies on malignant tumors . 30 adsorbent, blood cell - derived long chain DNA will be more It has been reported that circulating nucleic acids (DNA preferentially recovered than circulating DNA having a short RNA ) in whole blood become damaged , resulting in frag - chain length , whereby it is difficult to selectively recover mentation thereof when the circulating nucleic acids are circulating DNA . For this reason , circulating DNA has been released into peripheral blood from the nucleus, and conse conventionally recovered by carrying out a nucleic acid quently circulate as a mono - or poly -nucleosomes (structure 35 recovery method utilizing a nucleic acid adsorbent for in which DNA is wound around a nucleosome: histone plasma or serum where blood cell components had been octamer ) in peripheral blood . The length of circulating DNA previously removed from a whole blood sample . However , in whole blood has been reported to vary and has been there are problems that in this case a special apparatus such generally reported to be approximately 100 to 800 bp . as a centrifuge is necessary to recover plasma or serum and In particular, DNA in a mononucleosome has a very short 40 the recovery method cannot be carried out if not under a chain of 148 base pairs , and is short to an extent not well- equipped environment. comparable with the chain length of blood cell -derived Regarding a technique for concentrating short -chain genomic DNA in whole blood . It has been reported to be nucleic acids or a technique for separating short- chain difficult to recover mononucleosomal DNA due to such a nucleic acids from long - chain nucleic acids , for example , short chain length thereof, when compared with long - chain 45 Patent Document 3 ( Published Japanese Translation No . nucleic acids. 2011 -522529 of the PCT International Publication ) discloses Most of the conventional art methods for purifying a method in which a sample containing nucleic acids is nucleic acids are based on one of the following two prin - brought into contact with a nucleic acid adsorbent in the ciples . Many of the methods which have been used from presence of at least one chaotropic compound and 25 to 35 long ago are methods based on a one - step method where a 50 volume % of at least one of a branched alcohol and an sample containing nucleic acids, such as a whole blood unbranched alcohol, and then the nucleic acids adsorbed sample , is mixed with a buffer containing a chaotropic agent onto the nucleic acid adsorbent are eluted . Further, Patent and an organic extractant to thereby extract nucleic acids . At Document 4 ( PCT International Publication No. WO 2007 / least one of phenol and chloroform is mainly used as the 065934 ) discloses a method in which short - chain nucleic organic extractant . Impurities such as proteins are discarded 55 acids are preferentially adsorbed onto a nucleic acid adsor together with the organic phase , and the nucleic acids bent by a combined use of citrate rather than a chaotropic retained in the aqueous phase are isolated and recovered by salt , and an alcohol. a phase separation process. This method suffers from prob An object of the present invention is to provide a method lems in that toxic and harmful organic extractants must be of efficiently recovering short- chain DNA having a length of used and in that complicated processes are required for the 60 1000 bases or less, such as circulating DNA , directly from purification of nucleic acids from the aqueous phase recov - a whole blood sample without requiring serum separation or ered by the phase separation process . plasma separation , and a kit used in the same method . In view of the disadvantages of a one - step method , an alternative method to the one - step method has also been SUMMARY OF THE INVENTION established . The alternative method is a method based on 65 selective adsorption of nucleic acids onto a solid carrier A method of recovering short- chain nucleic acids, a (nucleic acid adsorbent) such as silicon dioxide. A sample method for purifying short- chain nucleic acids , and a kit for US 10 , 323 , 241 B2 recovering short -chain nucleic acids according to an aspect [ 10 ] The silicic acid - containing material may be silica , of the present invention are as follows. magnetic silica , glass, or silicon dioxide . [ 1 ] A method of recovering short - chain nucleic acids [ 11 ] The short -chain nucleic acid may be circulating according to a first aspect of the present invention includes : DNA . a step ( a ) of bringing a mixture having an alcohol concen - 5 [ 12 ] A method of purifying short -chain nucleic acids from tration of 0 to 5 volume % , the mixture being prepared by a whole blood sample according to a second aspect of the mixing a whole blood sample with a first buffer containing present invention uses the method of recovering short- chain one ormore members selected from the group consisting of nucleic acids according to the first aspect . a chaotropic agent and a surfactant, into contact with a first [ 13 ] A kit of recovering short - chain nucleic acids accord nucleic acid adsorbent to adsorb nucleic acids derived from 10 ing to a third aspect of the present invention is a kit used in the whole blood sample onto the first nucleic acid adsorbent, the method for recovering short- chain nucleic acids accord and then separating and removing the first nucleic acid ing to the first aspect, including : a first buffer which contains adsorbent to recover liquid components ; a step ( b ) of bring one or more members selected from the group consisting of ing a mixture having an alcohol concentration of 10 volume a chaotropic agent and a surfactant, and is mixed with a % or higher, the mixture being prepared by mixing the liquid 15 whole blood sample such that an alcohol concentration is 5 components recovered in the step ( a ) with a second buffer volume % or lower ; a second buffer which contains one or containing one or more members selected from the group more members selected from the group consisting of a consisting of a chaotropic agent and a surfactant and an chaotropic agent and a surfactant and an alcohol, and is alcohol, into contact with a second nucleic acid adsorbent to mixed with the liquid components from which the nucleic adsorb short -chain nucleic acids having a length of 1000 20 acid adsorbent was separated and removed after bringing the bases or less derived from the whole blood sample onto the nucleic acid adsorbent into contact with the mixture of the second nucleic acid adsorbent, and then separating and first buffer and the whole blood sample , such that an alcohol recovering the second nucleic acid adsorbent from the concentration is 10 volume % or higher ; and nucleic acid recovered liquid components , and a step (c ) of eluting and adsorbents . recovering nucleic acids from the second nucleic acid adsor - 25 [ 14 ] The kit for recovering short - chain nucleic acids bent recovered in the step ( b ) . according to the third aspect may be configured to recover [ 2 ] A nucleic acid having a length of 5000 bases or more circulating DNA from a whole blood sample . derived from the whole blood sample may be separated and According to the method of recovering short - chain removed by adsorbing the nucleic acid having a length of nucleic acids according to the first aspect of the present 5000 bases or more onto a nucleic acid adsorbent in the step 30 invention , short- chain nucleic acids can be efficiently recov ( a ) . ered without requiring a serum separation process or a [ 3 ] The first buffer may contain guanidine hydrochloride plasma separation process from a whole blood sample where and a surfactant, a concentration of guanidine hydrochloride long -chain nucleic acids such as blood cell - derived genomic may be higher than or equal to 1 mol/ L and lower than or DNA (typically , several tens of thousands to three hundred equal to a maximum solubility , and a concentration of the 35 million bp long ) and short - chain nucleic acids such as surfactant may be 1 to 10 volume % . circulating DNA ( 100 bp to 1000 bp long ) are present in [ 4 ] The alcohol may be isopropanol or ethanol, and an admixture . That is , according to the method of recovering alcohol concentration in the mixture of the liquid compo short - chain nucleic acids according to the first aspect of the nents recovered in the step (a ) and the second buffer in the present invention , efficient purification of short -chain step ( b ) may be 10 to 60 volume % . 40 nucleic acids such as circulating DNA from whole blood [5 ] The alcohol may be isopropanol, and an isopropanol samples can be achieved even in facilities without equip concentration in the mixture of the liquid components recov - ment such as a centrifuge necessary for a serum separation ered in the step ( a ) and the second buffer in the step (b ) may process or a plasma separation process. be 10 to 40 volume % . [ 6 ] The second buffer may contain one or more chaotropic 45 BRIEF DESCRIPTION OF THE DRAWINGS agents selected from the group consisting of guanidine thiocyanate and guanidine isothiocyanate and a surfactant, a FIG . 1 is a view showing the results of agarose electro concentration of the chaotropic agent may be higher than or phoresis of the recovered eluate in Reference Example 3 . equal to 0 . 5 mol/ L and lower than or equal to a maximum FIG . 2 is a view showing the results of agarose electro solubility , and a concentration of the surfactant may be 3 to 50 phoresis of the recovered eluate in Example 1 . 15 volume % . FIG . 3 is a view showing the results of agarose electro [ 7 ] The second buffer may contain 10 to 200 mmol/ L of phoresis of the recovered eluate in Example 2 . a cation salt . [ 8 ] The step (b ) may be a step (b ') of adding a proteolytic DETAILED DESCRIPTION OF THE enzyme to the liquid components recovered in the step ( a ) to 55 INVENTION carry out an enzymatic reaction , and a step ( b " ) of bringing the mixture of the liquid components and the second buffer into contact with the second nucleic acid adsorbent to adsorb A method of recovering short - chain nucleic acids accord short - chain nucleic acids having a length of 1000 bases or ing to an embodiment of the present invention (hereinafter , less derived from the whole blood sample onto the second 60 sometimes referred to as “ nucleic acid recovery method nucleic acid adsorbent, and then separating and recovering according to the present embodiment” ) is a method of the second nucleic acid adsorbent, after the step (b ') . recovering short -chain nucleic acids having a length of 1000 [ 9 ] The first nucleic acid adsorbent or the second nucleic bases or less from a whole blood sample based on the acid adsorbent may be one or more members selected from principle of recovering a nucleic acid by adsorbing the the group consisting of a silicic acid - containing material, 65 nucleic acid onto a nucleic acid adsorbent, and takes advan zeolite , aluminum oxide , titanium dioxide , zirconium diox - tage of the fact that the adsorbability of short -chain nucleic ide, kaolin , magnetic particles, ceramics, and polystyrene. acids onto a nucleic acid adsorbent is strongly influenced by US 10 , 323 , 241 B2 an alcohol concentration . The adsorbability of short - chain recovered by eluting the nucleic acid from the second nucleic acids onto a nucleic acid adsorbent is very weak nucleic acid adsorbent recovered in the step ( b ) . ( low ) under the environment of a low alcohol concentration With respect to adsorbability onto a nucleic acid adsor or the absence of an alcohol, and is strong ( high ) under the bent under the same environment, the adsorbability of high alcohol concentration environment. The adsorbability 5 long -chain nucleic acids is higher than adsorbability of short - chain nucleic acids . Therefore , in the case of being of long - chain nucleic acids onto a nucleic acid adsorbent is adsorbed onto a nucleic acid adsorbent in the presence of an also affected by alcohol concentration , but the effect of the alcohol by directly mixing a buffer containing an alcohol in alcohol concentration is smaller than for short -chain nucleic a whole blood sample , short - chain nucleic acids are hardly acids . Particularly in the case of very long nucleic acids such 10 contained in the nucleic acids recovered from the nucleic as genomic DNA , most nucleic acids are adsorbed onto a acid adsorbent, since long -chain nucleic acids are preferen nucleic acid adsorbent even under the environment of the tially adsorbed and short - chain nucleic acids are hardly absence of an alcohol. Therefore , in the nucleic acid recov adsorbed . In contrast , in the nucleic acid recovery method ery method according to the present embodiment, nucleic according to the present embodiment, long - chain nucleic acids adsorbed onto a nucleic acid adsorbentnt are Trstfirst 15 acids in the nucleic acids derived from a whole blood sample removed from a whole blood sample under the environment are firstly removed by adsorbing thereof onto a nucleic acid of a low alcohol concentration or the absence of an alcohol, adsorbent under the environment where the adsorbability of and then the remaining nucleic acids are recovered by short -chain nucleic acids is weak , and then the remaining adsorbing thereof onto the nucleic acid adsorbent under a nucleic acids not adsorbed onto the nucleic acid adsorbent high alcohol concentration environment. 20 are recovered by adsorbing thereof onto a fresh nucleic acid Specifically , the nucleic acid recovery method according adsorbent under the environment where short - chain nucleic to the present embodiment includes the following steps ( a ) acids can also be adsorbed . It is possible to efficiently to (c ). The mixture of a whole blood sample and a first buffer recover short- chain nucleic acids by adsorbing short -chain in the following step ( a ) does not contain an alcohol or has nucleic acids onto a nucleic acid adsorbent after removing an alcohol concentration of 5 volume % or lower, and the 25 most of long - chain nucleic acids from the whole blood alcohol concentration in the mixture of the liquid compo - sample -derived nucleic acids. nents recovered in the step ( a ) and a second buffer in the Although the present embodiment shows an example following step ( b ) is 10 volume % or higher . using two nucleic acid adsorbents , the nucleic acid recovery The nucleic acid recovery method according to the pres - may be carried out by a step in which using one nucleic acid ent embodiment includes : 30 adsorbent, long - chain nucleic acids are recovered , long ( 1 ) a step ( a ) of bringing the mixture of a whole blood chain nucleic acids adsorbed onto the nucleic acid adsorbent sample and a first buffer into contact with a first nucleic acid are eluted and the nucleic acid adsorbent is washed , and then adsorbent to thereby adsorb nucleic acids derived from the short - chain nucleic acids are adsorbed and recovered . whole blood sample onto the first nucleic acid adsorbent. The first buffer used in the present embodiment contains and then separating and removing the first nucleic acid 35 one or more members selected from the group consisting of adsorbent to recover liquid components , a chaotropic agent and a surfactant. The chaotropic agents to ( 2 ) a step (b ) of bringing the mixture of the liquid be contained may be used alone or in combination of two or components recovered in the step ( a ) and a second buffer more thereof . Similarly , the surfactants to be contained may into contact with a second nucleic acid adsorbent to adsorb be used alone or in combination of two ormore thereof. The a short- chain nucleic acid having a length of 1000 bases or 40 first buffer is preferably a buffer containing one or two or less derived from the whole blood sample onto the second more chaotropic agents and one or two or more surfactants . nucleic acid adsorbent, and then separating and recovering Examples of the chaotropic agent include a guanidine salt the second nucleic acid adsorbent from the recovered liquid such as guanidine hydrochloride (GHC1 ) , guanidine thio components , and cyanate (GTC ) or guanidine isothiocyanate (GITC ), sodium (3 ) a step ( c ) of eluting and recovering nucleic acids from 45 iodide , and sodium perchlorate . The chaotropic agent con the second nucleic acid adsorbent recovered in the step ( b ) . tained in the first buffer is preferably a guanidine salt and In the step ( a ) , short -chain nucleic acids are hardly more preferably guanidine hydrochloride. adsorbed and long - chain nucleic acids such as genomic The surfactant to be used may be suitably selected from DNA are preferentially adsorbed onto the first nucleic acid surfactants that are commonly used in the art when extract adsorbent, since the alcohol concentration in the mixture of 50 ing nucleic acids from biological samples . The surfactant a whole blood sample and a first buffer is low at 5 volume contained in the first buffer may be an ionic surfactant such % or lower ( inclusive of 0 volume % ). Therefore , by as sodium dodecyl sulfate (SDS ), but it is preferably a separating and removing the first nucleic acid adsorbent that non - ionic surfactant, more preferably Triton ( registered has adsorbed the nucleic acids, long - chain nucleic acids are trademark ) X ( Polyoxyethylene ( 10 ) Octylphenyl Ether ) , preferentially removed from whole blood sample -derived 55 Tween (registered trademark ) 20 (Polyoxyethylene (20 ) nucleic acids and consequently the abundance ratio of Sorbitan Monolaurate ), Tween ( registered trademark ) 40 short -chain nucleic acids in the remaining liquid compo - (Polyoxyethylene (20 ) Sorbitan Monopalmitate ), Tween nents becomes relatively high . (registered trademark ) 60 (Polyoxyethylene ( 20 ) Sorbitan Then , in the step ( b ) , the short- chain nucleic acids in the Monostearate ), Tween (registered trademark ) 80 (Polyoxy liquid components can be adsorbed onto a second nucleic 60 ethylene (20 ) Sorbitan Monooleate ), Nonidet ( registered acid adsorbentby bringing the recovered liquid components trademark ) P - 40 (Polyoxyethylene ( 9 ) Octylphenyl Ether) , into contact with the second nucleic acid adsorbent in a state Brij (registered trademark ) 35 (Polyoxyethylene (23 ) Lauryl of making an alcohol concentration to 10 volume % or Ether) , Brij ( registered trademark ) 58 (Polyoxyethylene (20 ) higher by mixing a second buffer into the recovered liquid Cethyl Ether) , digitonin or saponin , and still more preferably components . 65 Triton X , Tween 20 or Nonidet P - 40 . Then , in the step ( c ) , nucleic acids containing short -chain In the case where the first buffer contains a chaotropic nucleic acids derived from a whole blood sample can be agent, the concentration of the chaotropic agent may be any US 10 , 323 , 241 B2 concentration sufficient to extract nucleic acids from cells in Cethyl Ether ) , digitonin or saponin , and still more preferably a whole blood sample and may be appropriately adjusted in Triton X , Tween 20 or Nonidet P - 40 . consideration of the type of chaotropic agents to be used , In the case where the second buffer contains a chaotropic and whether or not chaotropic agents are used in combina - agent, the concentration of the chaotropic agent may be any tion with surfactants . For example , the concentration of the 5 concentration sufficient for the nucleic acid extracted from chaotropic agent in the first buffer is preferably higher than the cells by the first buffer to maintain the extracted state and or equal to 1 mol/ L and lower than or equal to the maximum solubility , and more preferably higher than or equal to 2 may be appropriately adjusted in consideration of the type of mol/ L and lower than or equal to the maximum solubility . chaotropic agents to be used , and whether or not chaotropic In the case where the first buffer contains a surfactant, the 10 agents are used in combination with surfactants . For concentration of the surfactant may be any concentration example , the concentration of the chaotropic agent in the sufficient to extract nucleic acids from cells in a whole blood second buffer is preferably higher than or equal to 0 . 5 mol/ L sample and may be appropriately adjusted in consideration and lower than or equal to the maximum solubility, more of the type of surfactants to be used , and whether or not preferably higher than or equal to 1 mol/ L and lower than or surfactants are used in combination with chaotropic agents . 15is equal toto the maximum solubility , and still more preferably For example , the concentration of the surfactant in the first higher than or equal to 2 mol /L and lower than or equal to buffer is preferably 1 to 10 volume % , and more preferably the maximum solubility . 2 to 5 volume % . In the case where the second buffer contains a surfactant, The first buffer is preferably a buffer which does not the concentration of the surfactant may be any concentration contain an alcohol, but may contain an alcohol. In the case 20 sufficient for the nucleic acid extracted from the cells by the where the first buffer contains an alcohol, the alcohol first buffer to maintain the extracted state and may be concentration in the first buffer and the mixing ratio of the appropriately adjusted in consideration of the type of sur first buffer and the whole blood sample are appropriately factants to be used , and whether or not surfactants are used adjusted such that the alcohol concentration in the mixture in combination with chaotropic agents . For example , the of the whole blood sample and the first buffer is 5 volume 25 concentration of the surfactant in the second buffer is % or lower. By adjusting the alcohol concentration in the preferably set to 3 to 15 volume % , and more preferably set mixture of the whole blood sample and the first buffer to 5 to 7 to 11 volume % . volume % or lower, the amount of short -chain nucleic acids The second buffer contains an alcohol. The alcohols having a length of 500 bases or less adsorbed onto the first contained in the second buffer may be used alone or in nucleic acid adsorbent separated and removed can be made 30 combination of two or more thereof. The alcohol to be used less than the detection limit value . The alcohols contained in is preferably branched or linear alkanols having 1 to 5 the first buffer may be used alone or in combination of two carbon atoms commonly used in the art when extracting or more thereof. The alcohol to be used is preferably purifying nucleic acids from biological samples . Examples branched or linear alkanols having 1 to 5 carbon atoms, of the alkanols include methanol, ethanol, propanol, isopro more preferably isopropanol or a mixed solvent of isopro - 35 panol, butanol and pentanol. The alcohol contained in the panol and ethanol , and still more preferably isopropanol. second buffer is preferably an alcohol having a nucleic acid The second buffer used in the present embodiment con - precipitation effect equivalent to isopropanol, more prefer tains one or more members selected from the group con a bly ethanol, isopropanol or a mixed solvent thereof, still sisting of a chaotropic agent and a surfactant. The chaotropic more preferably isopropanol or a mixed solvent of isopro agents to be contained may be used alone or in combination 40 panol and ethanol, and even more preferably isopropanol. of two or more thereof. Similarly , the surfactants to be The alcohol concentration in the second buffer, and the contained may be used alone or in combination of two or mixing ratio of the liquid components recovered in the step more thereof. The second buffer is preferably a buffer ( a ) and the second buffer are appropriately adjusted such that containing one or two or more chaotropic agents and one or the alcohol concentration in the mixture of the liquid com two or more surfactants . 45 ponents and the second buffer is 10 volume % or higher . In Examples of the chaotropic agent which may be contained the case where the alcohol contained in the second buffer is in the second buffer include the same compounds as the isopropanol, the isopropanol concentration in the second compounds which may be contained in the first buffer. The buffer and the mixing ratio of the liquid components and the chaotropic agent contained in the second buffer is preferably second buffer are preferably adjusted such that the isopro a guanidine salt , and more preferably guanidine thiocyanate 50 panol concentration in the mixture of the liquid components or guanidine isothiocyanate exerting strong chaotropic and the second buffer is 10 to 40 volume % . In the case effects even in a small amount thereofof. where the alcohol contained in the second buffer is ethanol, Examples of the surfactant which may be contained in the the ethanol concentration in the second buffer and the second buffer include the same compounds as the com - mixing ratio of the liquid components and the second buffer pounds which may be contained in the first buffer . The 55 are preferably adjusted such that the ethanol concentration in surfactant contained in the second buffer is preferably a the mixture of the liquid components and the second buffer non - ionic surfactant, more preferably Triton (registered is 30 to 60 volume % . trademark ) X (Polyoxyethylene ( 10 ) Octylphenyl Ether ) , The second buffer used in the present embodiment also Tween (registered trademark ) 20 (Polyoxyethylene ( 20 ) preferably further contains a cation salt . The cation salt may Sorbitan Monolaurate ) , Tween (registered trademark ) 40 60 be a monovalent cation salt or a divalent cation salt . (Polyoxyethylene (20 ) Sorbitan Monopalmitate ) , Tween Examples of the cation constituting the cation salt include a ( registered trademark ) 60 (Polyoxyethylene (20 ) Sorbitan lithium ion , a sodium ion , a potassium ion , a rubidium ion , Monostearate ), Tween (registered trademark ) 80 ( Polyoxy - a cesium ion , an ammonium ion , a magnesium ion , a ethylene ( 20 ) Sorbitan Monooleate ) , Nonidet ( registered calcium ion , and a strontium ion . The cation constituting the trademark ) P - 40 ( Polyoxyethylene ( 9 ) Octylphenyl Ether ), 65 cation salt is preferably a lithium ion , a sodium ion , a Brij ( registered trademark ) 35 ( Polyoxyethylene ( 23 ) Lauryl magnesium ion , or a calcium ion . The anion constituting the Ether ), Brij ( registered trademark ) 58 (Polyoxyethylene (20 ) anion salt is preferably a chloride ion , a bromide ion or an US 10 , 323 , 241 B2 10 iodide ion , more preferably a chloride ion or a bromide ion , the nucleic acid adsorbent. For example , in the case where and still more preferably a chloride ion . the first nucleic acid adsorbent is of a particulate shape, the In the case where the second buffer contains a cation salt, mixture of the whole blood sample and the first buffer can the cation salt concentration in the second buffer may be any be brought into contact with the nucleic acid adsorbent by concentration at which extraction / purification of nucleic 5 adding the nucleic acid adsorbent to the mixture . In the case acids is possible from a biological sample by an alcohol where the first nucleic acid adsorbent is fixed to the inner precipitation method , and is appropriately determined in wall surface of a container or the like , the mixture can be consideration of the type of cation salts to be used . With brought into contact with the nucleic acid adsorbent by respect to the second buffer used in the present embodiment, mixing the whole blood sample and the first buffer in the 10 to 200 mmol/ L is preferable taking into consideration that 10 container , or by placing the mixture of the whole blood the recovered short - chain nucleic acid is subjected to a sample and the first buffer in the container. commonly used analysis such as PCR . The whole blood sample - derived nucleic acids are The first buffer is prepared by dissolving at least one of a adsorbed onto a first nucleic acid adsorbent and then the first chaotropic salt and a surfactant, and if necessary , an alcohol nucleic acid adsorbent is separated and removed to recover and other ingredients in water or a buffer . The second buffer 15 liquid components . Separation and removal of the first is prepared by dissolving at least one of a chaotropic salt and nucleic acid adsorbent and recovery of the liquid compo a surfactant and an alcohol, and if necessary, a cation salt nents can be carried out by a widely used solid - liquid and other ingredients in water or a buffer . In the case where separation operation . For example , in the case where the first the solvent for preparing the first buffer and the second nucleic acid adsorbent is granular, only the liquid compo buffer is a buffer, a pH of the buffer is suitably determined 20 nents can be recovered by centrifuging a solution containing in consideration of the type of the nucleic acid to be the first nucleic acid adsorbent ( a mixture of the whole blood extracted (DNA or RNA ) , the analysis method where the sample , the first buffer and the first nucleic acid adsorbent ) recovered nucleic acid is used , and the like . In the present to precipitate the nucleic acid adsorbent, and then collecting embodiment, a pH value of the first buffer and the second the supernatant. Further, only the liquid components passed buffer is preferably pH 5 to 9 , and more preferably pH 6 . 5 25 through a porous membrane or a filter paper can also be to 8 .5 . In the case where the nucleic acid to be extracted is recovered by passing a solution containing the first nucleic DNA , a pH of 7 .0 to 8 . 5 is particularly preferable . The buffer acid adsorbent through a porous membrane or filter paper serving as the solvent for preparing the first buffer and the having a small pore diameter to such an extent that the second buffer may be appropriately selected from the buffers nucleic acid adsorbent cannot permeate . Additionally , in the commonly used in the art at the time of extraction , purifi- 30 case where the first nucleic acid adsorbent is magnetic cation and analysis of nucleic acids. Specific examples of the particles, only the liquid components can be recovered by buffer include a Tris ( trishydroxymethylaminomethane ) - using a magnet. In the case where the first nucleic acid HCl buffer, and a phosphate buffer. The water serving as the adsorbent is membrane - like such as a silica membrane , only solvent for preparing the first buffer and the second buffer is the liquid components can be recovered by permeating a preferably deionized water or ultrapure water. 35 mixture of the whole blood sample and the first buffer Further , the first buffer and the second buffer may contain through a silica membrane to thereby bring the mixture into other substances other than chaotropic agents , surfactants, contact with the nucleic acid adsorbent. When the mixture is alcohols and cation salts , as long as they do not inhibit the passed through a silica membrane, a liquid may be removed effect of the present embodiment. from the silica membrane by subjecting it to centrifugation , The first nucleic acid adsorbent and the second nucleic 40 vacuum or pressurization . acid adsorbent used in the present embodiment are not A first nucleic acid adsorbentmay be mixed with a whole particularly limited as long as they are a solid capable of blood sample simultaneously with mixing of the whole adsorbing nucleic acids . The material for the nucleic acid blood sample with a first buffer. Alternatively , the first adsorbent is preferably one or more members selected from nucleic acid adsorbentmay be brought into contact with the the group consisting of a silicic acid - containing material 45 mixture after sufficient extraction of nucleic acids from cells such as silica , magnetic silica , glass or silicon dioxide , in the whole blood sample by incubating the mixture of the zeolite , aluminum oxide , titanium dioxide , zirconium diox - whole blood sample and the first buffer for a certain period ide , kaolin , magnetic particles , ceramics and polystyrene , of time. Alternatively , liquid components may be immedi and more preferably a silicic acid - containing material. ately recovered by bring the first nucleic acid adsorbent into Among them , it is more preferable to use a silica membrane , 50 contact with the mixture of the whole blood sample and the silica gel, magnetic particles having silica or glass on the first buffer . Alternatively , the first nucleic acid adsorbent surface thereof, or a combination of these materials . The first may be separated and removed after incubating the mixture nucleic acid adsorbent and the second nucleic acid adsorbent of the whole blood sample and the first buffer for a certain may employ a material of the same type . period of time in a state of being in contact with the first The shape of the first nucleic acid adsorbent and the 55 nucleic acid adsorbent . second nucleic acid adsorbent is not particularly limited and In the step ( a ), the amount of the first nucleic acid may be of a particulate shape , a membrane shape , or a shape adsorbent to be used , and the time conditions until separat fixed to the inner wall surface of a container or the like . ing and removing the nucleic acid adsorbent from the time Hereinafter , individual steps will be described . of bringing the first nucleic acid adsorbent into contact with First, in the step ( a ) , a mixture of a whole blood sample 60 the mixture of the whole blood sample and the first buffer are and a first buffer is brought into contact with a first nucleic preferably adjusted such that almost all of the genomic DNA acid adsorbent. In the mixture of the whole blood sample derived from the whole blood sample is adsorbed onto the and the first buffer, nucleic acids are extracted from the cells first nucleic acid adsorbent, and the amount of the nucleic in the whole blood sample by the chaotropic agent or the acid having a length of several tens of thousand bases or surfactant derived from the first buffer . 65 more adsorbed onto the second nucleic acid adsorbent in the By bringing the mixture into contact with the first nucleic step ( b ) is less than the detection limit value . Further, it is acid adsorbent, the extracted nucleic acids are adsorbed onto more preferable to adjust such that almost all of the nucleic US 10 , 323 , 241 B2 11 acid having a length of 5000 bases or more derived from the with the second nucleic acid adsorbent. Specifically , the step whole blood sample are adsorbed onto the first nucleic acid (b ) is changed to the following steps ( b ') and ( b " ) . In the step adsorbent, and the amount of the nucleic acid having a (b " ) , the alcohol concentration in the mixture of the liquid length of 5000 bases or more adsorbed onto the second components and the second buffer after the enzymatic nucleic acid adsorbent in the step ( b ) is less than the 5 reaction is 10 volume % or higher . detection limit value . By removing a long -chain nucleic acid ( 1 ) a step ( b ') of adding a proteolytic enzyme to the liquid by sufficiently adsorbing thereof onto the first nucleic acid components recovered in the step ( a ) to carry out an enzy adsorbent in the step ( a ) , it is possible to selectively recover matic reaction , and a desired short - chain nucleic acid . (2 ) a step ( b " ) of bringing the mixture of the liquid The first buffer may contain a proteolytic enzyme. Use of 10 components and a second buffer into contact with a second a proteolytic enzyme enables to more efficiently carry out nucleic acid adsorbent to adsorb short -chain nucleic acids the extraction of nucleic acids from cells in a whole blood having a length of 1000 bases or less derived from the whole sample . Examples of the proteolytic enzyme include blood sample onto the second nucleic acid adsorbent , and lysozyme, proteinase , proteinase K , and cellulase . In the then separating and recovering the second nucleic acid case where the first buffer contains a proteolytic enzyme, it 15 adsorbent, after the step ( b ' ) . is preferable to carry out an enzymatic reaction by incubat- In the step ( b ' ) , a chaotropic agent or a surfactant may be ing the mixture of a whole blood sample and a first buffer for newly added together with a proteolytic enzyme. The chao approximately 5 minutes to 2 hours in a temperature range tropic agent and the surfactantmay employ the samemate where the contained proteolytic enzyme may exert enzy - rial as the material exemplified as a material which may be matic activity , preferably in the vicinity of an optimum 20 contained in a second buffer. In the case where the reaction temperature for the enzymatic activity of the proteolytic solution of the proteolytic enzyme reaction in the step (b ' ) enzyme. contains a chaotropic agent, the concentration of the chao The enzymatic reaction may be carried out prior to tropic agent in the reaction solution is preferably 0 . 5 to 2 .5 bringing a first nucleic acid adsorbent into contact with the mol/ L . In the case where the reaction solution of the mixture of the whole blood sample and the first buffer , or 25 proteolytic enzyme reaction in the step ( b ' ) contains a may be carried out after contacting therebetween and prior surfactant, the concentration of the surfactant in the reaction to separation and removal of the first nucleic acid adsorbent. solution is preferably 3 to 15 volume % , and more preferably Further , the incubation for the enzymatic reaction may also 7 to 11 volume % . serve as the incubation for adsorption of the extracted In the step ( b ') , a cation salt may be newly added together nucleic acid onto the first nucleic acid adsorbent. 30 with a proteolytic enzyme. The cation salt may employ the Then , as the step ( b ) , the mixture of the liquid components same material as the material exemplified as a material recovered in the step ( a ) and the second buffer is brought which may be contained in a second buffer . In the case where into contact with a second nucleic acid adsorbent to thereby the reaction solution of the proteolytic enzyme reaction in adsorb short -chain nucleic acids having a length of 1000 the step ( b ') contains a cation salt , the concentration of the bases or less derived from the whole blood sample onto the 35 cation salt in the reaction solution is preferably 10 to 200 second nucleic acid adsorbent , and then the second nucleic mmol/ L . acid adsorbent is separated and recovered from the liquid Then , as the step ( c ) , the nucleic acid is eluted and components . The method of bringing the mixture of the recovered from the second nucleic acid adsorbent recovered liquid components recovered in the step ( a ) and the second in the step (b ). The elution and recovery of the nucleic acid buffer into contact with the second nucleic acid adsorbent, or 40 from the second nucleic acid adsorbent may be carried out the method of adsorbing the nucleic acid in the liquid by a method which has been commonly carried out when components onto the second nucleic acid adsorbent may be eluting the nucleic acid adsorbed onto silica or the like . For carried out in the same manner as in the method of bringing example , water may be used as an eluting solution . Specifi the mixture of a whole blood sample and a first buffer into cally , by bringing water into contact with a second nucleic contact with a first nucleic acid adsorbent in the step ( a ), or 45 acid adsorbent, it is possible to elute the nucleic acid the method of adsorbing a nucleic acid derived from a whole adsorbed to the second nucleic acid adsorbent. blood sample onto a first nucleic acid adsorbent. Further , the The second nucleic acid adsorbent recovered in the step method of separating and recovering a second nucleic acid ( b ) may be washed with a suitable solution , prior to the step adsorbent may be carried out in the same manner as in the ( c ). From the viewpoint of no possibility of the nucleic acid separation and removal of a first nucleic acid adsorbent. 50 being desorbed from the nucleic acid adsorbent and also no In the case where a proteolytic enzyme was not contained incorporation of new salts or the like , the washing solution in the first buffer , it is also preferable to carry out a is preferably 70 to 95 volume % of an aqueous alcohol proteolytic enzyme reaction prior to separating and recov - solution , and more preferably 70 to 95 volume % of an ering a second nucleic acid adsorbent. The proteolytic aqueous ethanol solution . enzyme to be used may be the same material as the material 55 The whole blood sample subjected to the nucleic acid that may be contained in the first buffer. For example , it is recovery method according to the present embodiment may preferable to carry out an enzymatic reaction by containing be a sample containing blood cells and plasma components . a proteolytic enzyme in the second buffer and incubating a As the whole blood sample subjected to the nucleic acid mixture of the liquid components recovered in (a ) and a recovery method according to the present embodiment, it is second buffer for approximately 5 minutes to 2 hours in a 60 preferable to use whole blood collected from an animal such temperature range where the contained proteolytic enzyme as human or mouse as it is as a sample , but a sample can exert an enzymatic activity, preferably in the vicinity of subjected to a process that can leave at least a portion of an optimum temperature for the enzymatic activity of the blood cell components and plasma components in whole proteolytic enzyme . blood may be subjected to the step ( a ) as a whole blood In the case where carrying out a proteolytic enzyme 65 sample . An example of such a process may be a filtration reaction in the step ( b ) , it is also preferable to carry out the process which carries out size separation to remove or proteolytic enzyme reaction prior to bringing into contact recover only blood cell components of a particular size , or US 10 , 323 , 241 B2 13 14 specific adsorption separation to remove or recover only disease course of individuals affected by carcinomas , or for blood cell components presenting a specific surface antigen . observing the effects of anti - cancer therapies on malignant The nucleic acid recovered in the nucleic acid recovery tumors . method according to the present embodiment may be DNA Nucleic acid analysis including gene analysis usually or RNA . Further, the nucleic acid may be a single -stranded 5 analyzes a nucleic acid to be analyzed , and interprets the information obtained as a result of the analysis . The nucleic nucleic acid or a double - stranded nucleic acid and may be a acid analysis is primarily carried out by a method of directly linear nucleic acid or a circular nucleic acid such as a reading the base sequence of a nucleic acid by using a plasmid . Further , the nucleic acid may be an epigenetically sequencer or the like , a method using a polymerase reaction modified nucleic acid . Further , the nucleic acid may be a· 10 starting from a primer that specifically hybridizes to the nucleic acid contained in a whole blood sample , and the region consisting of a specific base sequence , or a method nucleic acid may be a cell nucleus - derived nucleic acid of using a probe that specifically hybridizes to the region cells of the organism species from which blood has been consisting of a specific base sequence . Since the short - chain collected , a mitochondria - derived nucleic acid , a nucleic nucleic acids obtained by the nucleic acid recovery method acid derived from a microorganism incorporated into such a 15 according to the present embodimentusually contain nucleic blood -collected organism , or an artificial or synthetic acids of a wide variety of genes or fragments thereof, it is nucleic acid . Examples of the artificial or synthetic nucleic preferable that the analysis target gene be analyzed by a acid include cosmids, vectors, cDNAs , and fragments method using at least one of a primer and a probe . Examples thereof. of the method using at least a primer include a PCR The nucleic acid recovery method according to the pres - 20 (polymerase chain reaction ) method , a real- time PCR ent embodiment can efficiently recover short- chain nucleic method , a TaqMan (registered trademark ) - PCR method , a acids having a length of 1000 bases or less . LAMP ( Loop -Mediated Isothermal Amplification ) method , With respect to the nucleic acid recovery method accord a SMAP ( SMart Amplification Process ) method , a NASBA ing to the present embodiment, a desired nucleic acid to be (Nucleic Acid Sequence -Based Amplification ) method , an recovered is preferably a short -chain nucleic acid having a 25 RCA ( rolling circle amplification ) method , and modified length of 800 bases or less , more preferably a short- chain methods thereof. Examples of the method using a probe nucleic acid having a length of 500 bases or less , or still include an Invader (registered trademark ) method , and a more preferably a short - chain nucleic acid having a length of DNA microarray . These methods may be used in combina 200 bases or less . The nucleic acid recovery method accord - tion as appropriate . ing to the present embodiment is suitable for concentrating 30 In the nucleic acid recovery method according to the and purifying circulating DNAs in whole blood , non -coding present embodiment, there is a case where merely a trace RNAs, miRNAs, short - chain synthetic nucleic acids, and the amount of short- chain nucleic acids is obtained . Therefore , like . the method for analyzing short - chain nucleic acids recov The nucleic acid recovery method according to the pres ered by the nucleic acid recovery method according to the ent embodiment is particularly suitable for purifying circu - 35 present embodiment is preferably ( 1 ) an analysis method lating DNAs in whole blood . In a conventional method , utilizing a nucleic acid amplification reaction using a probe , tumor -derived nucleic acids freely circulating in peripheral ( 2 ) a method including a first step of amplifying a nucleic blood have been purified and recovered from the serum or acid by PCR or the like , and a second step of carrying out plasma, whereas in the nucleic acid recovery method accord a nucleic acid analysis for the amplification product obtained ing to the present embodiment, freely circulating tumor - 40 in the first step . The above -mentioned method ( 1 ) may derived nucleic acids can be purified directly from a whole include , for example , an allele - specific extension method for blood sample , taking advantage of the selectivity of a carrying out an extension reaction using a primer specific for nucleic acid to be adsorbed onto a nucleic acid adsorbent. In a particular genotype , and TaqMan - PCR method . The addition , the nucleic acid recovery method according to the above- mentioned method ( 2 ) may include , for example , an present embodiment can also be automated , since the opera - 45 Invader Plus method , a restriction fragment length polymor tion of individual steps is relatively simple . Further , circu phism (PCR -RFLP ) method , and a single strand conforma lating DNAs recovered by the nucleic acid recovery method tion polymorphism ( PCR - SSCP ) method . Among them , an according to the present embodiment can be suitably sub - Invader Plus method is preferable , in which the short - chain jected to a variety of analysis methods, since those circu - nucleic acid obtained by the nucleic acid recovery method lating DNAs recovered have been purified so as to remove 50 according to the present embodiment is first amplified by long -chain nucleic acids such as genomic DNA . That is , the PCR , and then an Invader method is carried out on the nucleic acid recovery method according to the present obtained amplification product because it is possible to carry embodiment is a method that can be developable in the out a nucleic acid analysis with high sensitivity and high clinical research and diagnostic field by combining various accuracy even in the case where the short- chain nucleic acid analysis methods . 55 to be analyzed is present in a very small amount. Further, it The short -chain nucleic acid obtained by the nucleic acid is also possible to employ a nucleic acid analysis method recovery method according to the present embodiment may that does not require nucleic acid amplification steps such as be subjected to any nucleic acid analysis , but it is preferable PCR . to be subjected to the analysis of particularly gene polymor - Hereinafter, an example of a method to detect and analyze phism and gene mutation ( genetic test or the like ) and it is 60 a particular gene ( gene to be analyzed ) by an Invader Plus more preferable to be subjected to the analysis of SNP method is shown , but the nucleic acid analysis method of ( single nucleotide polymorphism ) and single gene mutation . short -chain nucleic acids recovered by the nucleic acid In particular, in the case where circulating DNA derived recovery method according to the present embodiment is not from cancer cells is intended to be a short - chain nucleic acid limited thereto . to be recovered , the recovered circulating DNA is subjected 65 First, an Invader Plus buffer which is an Invader Plus to a nucleic acid analysis for various purposes , such as for reaction solution , an oligo mix for detecting a gene to be assessing the prognosis of carcinomas , for monitoring the analyzed , and an enzymemix were mixed in the short - chain US 10 , 323 , 241 B2 15 16 nucleic acid solution recovered by the nucleic acid recovery examined for the adsorbability thereof to a silica membrane method according to the present embodiment ( in the step ( c ) , filter ( corresponding to a nucleic acid adsorbent ) in a state of an eluate in which nucleic acids were eluted from a second those samples being dissolved in buffer 1( 3 mol /L of guani nucleic acid adsorbent) , followed by denaturation (heating at dine hydrochloride, and 10 volume % of Triton X ) or buffer 95° C . for 2 minutes ) to melt double helix of DNA into 5 2 ( 1 . 2 mol / L of guanidine thiocyanate , 10 volume % of single strands. The oligo mix includes PCR primers , DNA Tween20 , 40 volume % of isopropanol, and 100 mmol/ L of probes, invading oligos, and a FRET cassette . MgC1, ). Note that buffer 1 corresponds to a first buffer used Then , PCR is carried out, resulting in an amplification in the nucleic acid recovery method according to a first reaction of a nucleic acid of a portion corresponding to the embodiment of the present invention , and buffer 2 corre gene to be analyzed , and then deactivation of polymerase is 10 sponds to a second buffer used in the nucleic acid recovery carried out to stop PCR . method according to the present embodiment. Finally , an Invader ( registered trademark ) method is car- Specifically , a DNA solution obtained by dissolving 1000 ried out to degrade a DNA probe which has specifically n g of DNA in buffer 1 or buffer 2 was passed through a silica hybridized to the region having a nucleic acid sequence membrane filter to thereby adsorb the DNA onto the silica corresponding to the gene to be analyzed . Then , the 5 '- oli - 15 membrane filter . Then , purified water was passed through gonucleotide of the heated probe of DNA produced by the the silica membrane filter to elute the DNA from the silica decomposition reaction is combined with an FRET cassette membrane filter. having a fluorescent dye . The FRET cassette is degraded and The DNA concentration in the resulting eluate was mea a fluorescent dye is liberated from the FRET cassette , sured using a PicoGreen reagent ( Invitrogen ) to calculate the resulting in emission of fluorescence . The gene to be ana - 20 total amount of DNA in the eluate ( recovered amount of lyzed can be indirectly detected and measured by measuring DNA ) . The total amount of DNA in the eluate corresponds this fluorescence. In other words, the fluorescence signal to an amount adsorbed onto the silica membrane filter . From intensity indicates a detected amount of the corresponding the calculated total DNA amount in the eluate and the total gene to be analyzed . DNA amount in the DNA solution ( 1000 ng ) which had been The fluorescent signal intensity of a fluorescent dye 25 passed through a silica membrane filter, the DNA recovery dissociated by an Invader ( registered trademark ) method (for rate ( % ) using a silica membrane filter was calculated example , the fluorescent signal intensity ( excitation wave ( calculation equation : [ Total DNA amount in eluate (ng ) ] / length : 485 nm , and emission wavelength : 535 nm ) of FAM [ 1000 (ng ) ]x100 ). The calculated recovery rate is shown in in the case where the fluorescent dye is FAM ) is measured Table 1 . using a fluorometer . The fluorometer may be , for example , 30 a fluorometer LightCycler480 manufactured by Roche TABLE 1 Applied Science. < Kit of Recovering Short -Chain Nucleic Acid > DNA recovery The nucleic acid recovery method according to a second embodiment of the present invention can be more conve - 35 DNA length Buffer 1 Buffer 2 niently carried out by fabricating reagents used , and the like 200 bp 6 . 5 % 89 . 8 % into a kit. The kit for carrying out the nucleic acid recovery 500 bp 6 . 2 % 84 . 4 % method according to the present embodiment preferably 1000 bp 29 . 4 % 89 . 3 % contains the first buffer , the second buffer , and nucleic acid Genomic DNA 85 . 1 % 91. 8 % adsorbent used as the first nucleic acid adsorbent and the 40 second nucleic acid adsorbent. The first buffer is a buffer As shown in Table 1 , in the case of DNA dissolved in which is mixed with a whole blood sample such that the buffer 2 where an isopropyl alcohol concentration is 40 alcohol concentration in the mixture is 5 volume % or lower. volume % , the DNA recovery rate was very high at 80 % or The second buffer is a buffer which is mixed in the liquid higher regardless of the DNA length . The DNA recovery rate components obtained by bringing a nucleic acid adsorbent 45 in the case of DNA dissolved in buffer 1 containing no into contact with the mixture of the first buffer and the whole alcohol was high at 80 % or higher in genomic DNA , but it blood sample , and then separating and removing the nucleic was merely approximately 30 % in dsDNA (double - stranded acid adsorbent, such that the alcohol concentration in the DNA ) of 1000 bp , very low with less than 10 % in dsDNA mixture is 10 volume % or higher. Additionally , the kit may of 500 bp and 200 bp , and short - chain nucleic acids of 1000 contain a proteolytic enzyme for adding to a first buffer or 50 bp or less were found to exhibit almost no adsorption onto the like , a washing solution for washing a second nucleic a silica membrane filter . From these results , it was found that acid adsorbent onto which nucleic acids are adsorbed , an short -chain nucleic acids of 1000 bp or less are hardly eluate for eluting nucleic acids from a second nucleic acid adsorbed and long- chain nucleic acids such as blood cell adsorbent onto which nucleic acids are adsorbed , or the like , derived genomic DNA are preferentially adsorbed in the if necessary 55 absence of an alcohol when adsorbing nucleic acids onto a nucleic acid adsorbent. Further, from these results , it was EXAMPLES found that under the environment where an isopropanol concentration is 40 volume % , short- chain nucleic acids of Hereinafter , the present invention will be described in 1000 bp or less , particularly 800 bp or less are also strongly detail with reference to the following examples , but the 60 adsorbed onto a nucleic acid adsorbent and then can be present invention is not limited thereto . recovered . From these results , it has been suggested that short- chain nucleic acids of 1000 bp or less including Reference Example 1 circulating DNA, particularly short- chain nucleic acids of 800 bp or less can be recovered by first adsorbing whole dsDNA fragment standard samples of 200 bp , 500 bp and 65 blood sample - derived nucleic acids onto a nucleic acid 1000 bp (manufactured by Gensura Laboratories ) , and a adsorbent in the absence of an alcohol to be able to selec human genomic DNA (manufactured by GEN Script) were tively remove long - chain nucleic acids while leaving short US 10 , 323 , 241 B2 17 18 chain nucleic acids of 1000 bp or less in liquid components , using buffers 3 and 4 ( hereinafter , referred to as “ step and then by bringing a fresh nucleic acid adsorbent into ( b ' ) + ( b " ) variant” ) , using a commercially available nucleic contact with the remaining liquid components freed of thethe acid purification kit QIAamp Circulating Nucleic Acid Kit nucleic acid adsorbent in the presence of a high - concentra - (manufactured by Qiagen ) according to the instructions tion alcohol to thereby adsorb nucleic acids. 5 attached to the kit . The amount of DNA in the recovered The pH of each buffer in the embodiment of the present DNA solution (recovered amount of DNA ), the DNA recov invention was between 7 .5 and 8 .0 . ery amount per 1 mL of human plasma or human serum Reference Example 2 (ng / sample 1 mL ) , and the purity of DNA ( [ A260 ] / [A280 ] ) 10 were determined in the same manner as in the eluate Commercially available human plasma and human serum recovered in the step ( b ' ) + ( b " ) variant. (both manufactured by Lonza ) were subjected to an enzy Each measurement was carried out independently in trip matic treatment with a proteolytic enzyme, and then short licate , and an average and standard deviation were deter chain nucleic acids were recovered . Proteinase K (Merck ) mined . The results are shown in Table 2 . Consequently , in was used as a proteolytic enzyme. is the case where nucleic acids were recovered from human Specifically , first, 500 uL of proteinase K and 5 mL of plasma, the recovery amount of DNA and the purity of buffer 3 ( 2 . 0 mol/ L of guanidine thiocyanate , and 15 volume recovered DNA were both comparable in the step ( b ') + ( b " ) % of Tween20 ) were added to 5 mL of human plasma or variant and the method using a commercially available human serum , followed by mixing to prepare an enzymatic nucleic acid purification kit. In addition , in the case where reaction solution . The resulting enzymatic reaction solution 20 nucleic acids were recovered from human serum , the step was incubated at 52° C . for 30 minutes to carry out an ( b ') + (b " ) variant was able to recover a high -purity DNA enzymatic reaction , followed by heating at 70° C . for 10 comparable to that of the method using a commercially minutes to inactivate proteinase K . available nucleic acid purification kit in a larger amount than Then , 10 mL of buffer 4 ( 1 . 5 mol/ L of guanidine thio the method using a commercially available nucleic acid cyanate , 10 volume % of Tween20 , 40 volume % of iso purification kit . TABLE 2 Recovered amount Recovered of DNA per 1 mL amount Purity of DNA of sample (ng DNA Recovery of DNA ( ng ) ( A260 / A280 ) sample 1 mL ) sample method Average S . D . Average S . D . Average Human Step (b ') + ( b " ) 66. 5 1. 8 0 .9 0 .2 133 plasma variant Commercially 63 .5 2 . 2 1 . 0 0 . 1 127 available nucleic acid purification kit Human Step ( b' ) + (b ") 103. 1 11. 6 1. 1 0 .0 206 serum variant Commercially 61. 4 11. 7 0. 9 0. 0 123 available nucleic acid propanol, and 100 mmol/ L of MgCl2 ) was added to the 45 Reference Example 3 post - inactivation reaction solution , followed by mixing. Note that buffer 4 corresponds to the second buffer used in Nucleic acids in the human whole blood were recovered the nucleic acid recovery method according to a first using a first buffer and a silica membrane filter (nucleic acid embodiment of the present invention , and the alcohol con - adsorbent) used in the nucleic acid recovery method accord centration of the resulting mixture was approximately 20 50 ing to a first embodiment of the present invention . Buffer 1 volume % . The mixture was passed through a silica mem - used in Reference Example 1 was employed as the first brane filter to adsorb DNA on the silica membrane filter . buffer . Thereafter , a 70 mass % aqueous ethanol solution was Specifically , a mixture of 100 uL of human whole blood permeated through the silica membrane filter to wash the and 500 uL of buffer 1 was passed through a silica mem filter, and then purified water was passed to elute the DNA 55 brane filter to adsorb human whole blood - derived nucleic from the silica membrane filter. acids onto the silica membrane filter. Then , a 70 (mass / The amount of DNA in the resulting eluate ( recovered volume) % aqueous ethanol solution was permeated through amount of DNA ) was determined in the same manner as in the silica membrane filter to wash the filter , and purified Reference Example 1 , and the recovered amount of DNA per water was passed to elute nucleic acids from the silica 1 mL of human plasma or human serum (DNA recovery 60 membrane filter. amount per 1 mL of a sample (ng / sample 1 mL ) ) was The chain length of DNA contained in the resulting eluate calculated . Further, the ratio of absorbance at 260 nm was confirmed by agarose electrophoresis . FIG . 1 shows the ( A260 ) vs. absorbance at 280 nm ( A280 ) of the eluate electrophoresis results . Referring to FIG . 1 , the left lane (M ) ( [ A260 ] / [ A280 ] ) was determined in order to examine the is the electrophoresis results of a 1 kbp Ladder DNA marker, purity of recovered DNA . 65 and the right lane ( S ) is the electrophoresis results of the As a control , nucleic acids were purified from the same eluate . As shown in FIG . 1 , the eluate contained merely amount of human plasma or human serum as in the method DNA of a long chain length , and particularly a band smaller US 10 , 323 , 241 B2 19 20 than a 1 kbp marker was not observed at all . From these according to a first embodiment of the present invention . In results , it was demonstrated that long - chain nucleic acids are addition , from the human serum sample which was prepared selectively adsorbed and short- chain nucleic acids are not the same donor- derived human whole blood having an equal adsorbed in the case where nucleic acids are adsorbed by a volume of the nucleic acid -recovered human whole blood nucleic acid adsorbent from the mixture of the first buffer 5 sample , short - chain nucleic acids were recovered by the and the whole blood sample used in the nucleic acid recov steps ( b ) and ( c ) in the nucleic acid recovery method ery method according to the present embodiment. That is , it according to the present embodiment. was found that there is no risk of unintentional recovery of For recovering nucleic acids from a human whole blood short- chain nucleic acids including circular DNA in the step sample , specifically , 1 mL of a human whole blood sample (a ) of the nucleic acid recovery method according to the 10 and 5 mL of buffer 1 used in Reference Example 1 were present embodiment. mixed , and the prepared mixture was passed through a silica membrane filter. 600 uL of proteinase K and 6 mL of buffer Reference Example 4 3 used in Reference Example 2 were added to 5 mL of the The effect of alcohol concentration on the strength of solution ( liquid components ) permeated through the silica DNA of 200 bp , 500 bp or 1000 bp to adsorb onto a nucleic 15 membrane filter, followed by mixing to prepare an enzy acid adsorbent was examined . dsDNA fragment standard matic reaction solution . The resulting enzymatic reaction samples of 200bp , 500 bp and 1000 bp used in Reference solution was incubated at 52° C . for 30 minutes to carry out Example 1 were employed as the dsDNA . In addition , an enzymatic reaction , followed by heating at 70° C . for 10 alcohol- free buffer 4 - 1 ( 3 mol/ L of guanidine hydrochloride, minutes to inactivate proteinase K . and 10 volume % of Triton X ), 5volume % alcohol- con - 20 Then , 12 mL of buffer 4 used in Reference Example 2 was taining buffer 4 - 2 ( 3 mol/ L of guanidine hydrochloride , 10 added to the post- inactivation reaction solution , followed by volume % of Triton X , and 5 volume % of isopropanol ) , and mixing . The alcohol concentration of the resulting mixture 10 volume % alcohol- containing buffer 3 ( 3 mol/ L of was approximately 20 volume % . The mixture was passed guanidine hydrochloride , 10 volume % of Triton X , and 10 through a silica membrane filter to adsorb DNA on the silica volume % of isopropanol) were used as a buffer for dissolv - 25 membrane filter . Thereafter, 70 (mass / volume) % of an aque ing DNA . ous ethanol solution was permeated through the silica mem Specifically , a DNA solution where 1000 ng of DNA had brabrane filter to wash the filter, and then purified water was been dissolved in buffer 4 - 1 , 4 - 2 or 4 - 3 was passed through passed to elute the DNA from the silica membrane filter. a silica membrane filter to adsorb the DNA onto the silica For recovering nucleic acids from a human serum sample , membrane filter . Then , purified water was passed through 30 specifically, 50 uL of proteinase K and 500 uL of buffer 3 the silica membrane filter to elute the DNA from the silica used in Reference Example 2 were added to a human serum membrane filter. sample (500 ?L ) prepared from 1 mL of a human whole The total amount of DNA in the resulting eluate ( recov blood sample , followed by mixing to prepare an enzymatic ered amount of DNA ) and the DNA recovery rate ( % ) were reaction solution . In the same manner as in the recovery of determined in the same manner as in Reference Example 1 . 35 nucleic acids from a human whole blood sample , the result The results are shown in Table 3 . Consequently , DNA of 200 ing enzymatic reaction solution was subjected to an enzy bp and 500 bp could hardly be recovered in the case of using matic reaction , and then a mixture where buffer 4 had been buffer 4 -2 where the isopropanol concentration is 5 volume added to the inactivated reaction solution was passed % , similar to the case of using buffer 4 - 1 where the isopro through a silica membrane filter to elute the nucleic acids panol concentration is o volume % . To the contrary , in the 40 adsorbed onto the silica membrane filter. case of using buffer 4 - 3 where the isopropanol concentration The amount of DNA in the resulting eluate ( recovered is 10 volume % , the recovery rate of DNA of 200 bp have amount of DNA ) and the purity of DNA ([ A260 ]/ [A280 ] ) remarkably increased by 20 % or higher, and the recovery were determined in the same manner as in Reference rate of DNA of 500 bp have remarkably increased by 50 % Example 2 . The results are shown in Table 4 . As shown in or higher. Based on these results , it has been suggested that 45 Table 4 . the DNA recovery amount recovered from the the loss of short - chain nucleic acids can be inhibited by human whole blood sample was almost comparable to the adjusting the alcohol concentration in the mixture of a first amount recovered from the human serum sample . Based on buffer and a whole blood sample to be 5 volume % or lower, these results , it has been suggested that short - chain nucleic in the case where the first buffer contains an alcohol in the acids including desired circulating DNA can be selectively nucleic acid recovery method according to a first embodi - 50 recovered from the whole blood sample according to the ment of the present invention . nucleic acid recovery method according to the present embodiment, by selectively removing long - chain nucleic TABLE 3 acids such as blood cell -derived genomic DNA . DNA recovery 55 TABLE 4 Buffer 4 - 1 Buffer 4 - 2 Buffer 4 - 3 ( Alcohol ( Alcohol ( Alcohol Recovered amount of DNA Purity of DNA DNA length o volume % ) 5 volume % ) 10 volume % ) Sample name (ng ) (A260 / A280 ) 200 bp 6 . 5 % 8 . 9 % 20 . 5 % Human whole blood 120 . 0 0 . 7 500 bp 6 . 2 % 10 . 3 % 58 . 3 % 60 sample 1000 bp 29 . 4 % 34 . 0 % 75 . 2 % Human serum sample 117 . 5 1 . 0 In the samemanner as in Reference Example 3 , the chain Example 1 length of DNA obtained from a human whole blood sample 65 by the nucleic acid recovery method according to the present A short - chain nucleic acid was recovered from a human embodiment was confirmed by acrylamide gel electropho whole blood sample by the nucleic acid recovery method resis . FIG . 2 shows the electrophoresis results . Referring to US 10 , 323 , 241 B2 21 22 FIG . 2 , the left lane ( M ) is the electrophoresis results of a That is , the importance of the treatment previously 100 bp Ladder DNA marker, and the right lane ( S ) is the removing long - chain nucleic acids such as blood cell - de electrophoresis results of the eluate . As shown in FIG . 2 , the rived genomic DNA has been suggested in the step ( a ) of the eluate contained merely fragmented DNA of a short chain nucleic acid recovery method according to the present length , and particularly a band larger than a 1000 bp marker 5 embodiment. was not observed at all. Further, fragmentation of DNA could be confirmed from the fact that the band has been Example 3 smeared . Based on these results , it has been suggested that short A short -chain nucleic acid was recovered from a human chain nucleic acids including desired circulating DNA can 10 whole blood sample by the nucleic acid recovery method be selectively recovered from the whole blood sample according to a first embodiment of the present invention . according to the nucleic acid recovery method according to For recovering nucleic acids from a human whole blood the present embodiment, by selectively removing long- chain sample , specifically DNA was eluted in the same procedure nucleic acids such as blood cell -derived genomic DNA . as in Example 1 using buffers 5 and 6 where ethanol was 15 mixed in place of isopropanol in buffers 3 and 4 used in Example 2 Reference Example 2 . The amount of DNA in the resulting eluate (recovered A short- chain nucleic acid was recovered from a human amount of DNA ) and the purity of DNA ( [A260 ]/ [A280 ] ) whole blood sample by the steps ( b ) and ( c ) in the nucleic were determined in the same manner as in Reference acid recovery method according to a first embodiment of the 20 Example 2 . The results are shown in Table 6 . As shown in present invention . That is , in Example 2 , a short - chain Table 6 , it can be seen that the DNA recovery amount nucleic acid was recovered by only the steps ( b ) and ( c ) in recovered from the human whole blood sample was slightly the nucleic acid recovery method according to the present less , as compared to the results of Example 1 using isopro embodiment, and the step ( a ) was not done. panol ( Table 4 ) . For recovering nucleic acids from a human whole blood 25 Based on these results , it is clear that short -chain nucleic sample , specifically , 50 uL of proteinase K and 500 uL of acids including desired circulating DNA can be selectively buffer 3 used in Reference Example 2 were added to 500 uL recovered from the whole blood sample by selectively of a human whole blood sample ( the same sample as in removing long- chain nucleic acids such as blood cell - de Example 1 ) , followed by mixing to prepare an enzymatic rived genomic DNA , even in the case of using ethanol as a reaction solution . In the same manner as in the recovery of 30 substitute for isopropanol in the nucleic acid recovery nucleic acids from a human whole blood sample, the result - method according to the present embodiment. It is clear that ing enzymatic reaction solution was subjected to an enzy - the case of using isopropanol is more preferable than the matic reaction , and then a mixture where buffer 4 had been case of using ethanol in the nucleic acid recovery method added to the inactivated reaction solution was passed according to the present embodiment. through a silica membrane filter to elute the nucleic acids 35 adsorbed onto the silica membrane filter. TABLE 6 The amount of DNA in the resulting eluate (recovered amount of DNA ) and the purity of DNA ( A2601/ [ A2801) Recovered amount of DNA Purity of DNA were determined in the same manner asand in Reference Sample name (ng ) (A260 /A280 ) Example 2 . The results are shown in Table 5 . As shown in 40 Human whole blood 103 . 3 0 . 8 Table 5 , it can be seen that the DNA recovery amount sample recovered from the human whole blood sample was signifi cantly higher than the recovered amount of DNA from a human serum sample in Example 1 ( Table 4 ) . Example 4 45 TABLE 5 From a human whole blood sample , a short - chain nucleic acid was recovered without performing an enzymatic treat Recovered amount of DNA Purity of DNA ment ( a protease treatment ) in the nucleic acid recovery Sample name ( ng ) ( A260 /A280 ) method according to a first embodiment of the present Human whole blood 1600 . 0 2 . 1 50 invention . sample For recovering nucleic acids from a human whole blood sample , specifically DNA was eluted in the same procedure In the samemanner as in Reference Example 3 , the chain as in Example 1 using buffers 1 and 2 used in Reference length of DNA obtained from a human whole blood sample Example 1, except that a protease treatment was not carried was confirmed by agarose gel electrophoresis . FIG . 3 shows 55 out. the electrophoresis results . Referring to FIG . 3 , the left lane The amount of DNA in the resulting eluate (recovered ( M ) is the electrophoresis results of a 1 kbp Ladder DNA amount of DNA ) and the purity of DNA ( [ A2601/ [ A2801) marker , and the right lane ( S ) is the electrophoresis results were determined in the same manner as in Reference of the eluate . As shown in FIG . 3 , the eluate contained Example 2 . The results are shown in Table 7 . As shown in merely DNA of a long chain length , and particularly a band 60 Table 7 , it can be seen that the DNA recovery amount smaller than a 1 kbp marker was not observed at all. recovered from the human whole blood sample was less but From these results , it is clear that long - chain nucleic acids slightly, as compared to the results of Example 1 ( Table 4 ) . are preferentially adsorbed and short - chain nucleic acids are based on these results , it has been suggested that short not adsorbed in the case where short - chain nucleic acids are chain nucleic acids including desired circulating DNA can recovered by the steps (b ) and ( c) (without doing the step 65 be selectively recovered from the whole blood sample by ( a ) ) in the nucleic acid recovery method according to the selectively removing long - chain nucleic acids such as blood present embodiment. cell -derived genomic DNA , even in the case without a US 10 , 323 , 241 B2 23 24 protease treatment in the nucleic acid recovery method each surfactant of the first surfactant contained in the according to the present embodiment . first buffer and the second surfactant contained in the second buffer includes an ionic surfactant including TABLE 7 sodium dodecyl sulfate or at least one non - ionic surfactant having a polyoxyethylene group , the at Recovered amount of DNA Purity of DNA least one non - ionic surfactant selected from the Sample name (ng ) (A260 /A280 ) group consisting of polyoxyethylene ( 10 ) octylphe Human whole blood 99 . 6 0 . 5 nyl ether, polyoxyethylene ( 20 ) sorbitan monolau sar rate , polyoxyethylene (20 ) sorbitan monopalmitate , 10 polyoxyethylene ( 20 ) sorbitan monostearate , poly oxyethylene (20 ) sorbitan monooleate , polyoxyeth ylene ( 9 ) octylphenyl ether, polyoxyethylene ( 23 ) What is claimed is : lauryl ether , polyoxyethylene (20 ) cetyl ether, digi 1 . A method of recovering short- chain nucleic acids tonin , and saponin , and having a length of 1000 bases or less from a whole blood 15 each absorbent of the first nucleic acid adsorbent and sample , comprising : the second nucleic acid adsorbent includes at least a step (a ) of one selected from the group consisting of a silicic bringing a first mixture into contact with a first nucleic acid - containing material , zeolite , aluminum oxide , acid adsorbent, the first mixture having an alcohol titanium dioxide , zirconium dioxide, kaolin , mag concentration of 0 to 5 volume % , the first mixture 20 netic particles, ceramics , and polystyrene . being prepared by mixing a whole blood sample with 2 . The method of recovering short- chain nucleic acids a first buffer containing at least one selected from a according to claim 1 , wherein , in the step ( a ) , a nucleic acid first chaotropic agent and a first surfactant, the first having a length of 5000 bases or more derived from the nucleic acid adsorbent to adsorb nucleic acids whole blood sample is to be separated and removed from the derived from the whole blood sample onto the first 25 first mixture by the first nucleic acid adsorbent adsorbing the nucleic acid adsorbent, and then nucleic acid having the length of 5000 bases or more onto separating and removing the first nucleic acid adsorbent the first nucleic acid adsorbent. from being in contact with the first mixture , to 3 . The method of recovering short - chain nucleic acids recover liquid components of the first mixture ; according to claim 1 , wherein a step ( b ) of 30 the first buffer contains guanidine hydrochloride and the bringing a second mixture into contact with a second first surfactant, nucleic acid adsorbent, the second mixture having an a concentration of the guanidine hydrochloride in the first alcohol concentration of 10 volume % or higher, the buffer is higher than or equal to 1 mol/ L and lower than second mixture being prepared by mixing the liquid or equal to a maximum solubility of the guanidine components recovered in the step ( a ) with a second 35 hydrochloride in the first buffer , and buffer containing an alcohol, a cationic salt, and at a concentration of the first surfactant is 1 to 10 volume % . least one selected from a second chaotropic agent 4 . The method of recovering short - chain nucleic acids and a second surfactant, the second nucleic acid according to claim 1 , wherein adsorbent to adsorb short -chain nucleic acids having each alcohol of the alcohol in the first mixture when the a length of 1000 bases or less, the short - chain nucleic 40 first mixture includes the alcohol and the alcohol in the acids derived from the whole blood sample onto the second mixture is isopropanol or ethanol, and second nucleic acid adsorbent , and then an alcohol concentration in the second mixture of- pre separating and recovering the second nucleic acid pared by mixing the liquid components recovered in the adsorbent from being in contact with the second step ( a ) with the second buffer is 10 to 60 volume % . mixture including the recovered liquid components ; 45 5 . The method of recovering short -chain nucleic acids and according to claim 1 , wherein a step ( c ) of each alcohol of the alcohol in the first mixture when the eluting the short - chain nucleic acids absorbed in the first mixture includes the alcohol and the alcohol in the second nucleic acid adsorbent by bringing water into second mixture is isopropanol, and contact with the second nucleic acid adsorbent 50 an isopropanol concentration in the second mixture pre recovered in the step ( b ) and recovering the short pared by mixing of the liquid components recovered in chain nucleic acids from the second nucleic acid the step ( a ) with the second buffer is 10 to 40 volume adsorbent, % . wherein 6 . The method of recovering short - chain nucleic acids when the first buffer contains the alcohol, the alcohol 55 according to claim 1 , wherein contained in the first buffer is at least one selected the second buffer contains guanidine thiocyanate as the from branched or linear alkanols respectively having second chaotropic agent and the second surfactant, 1 to 5 carbon atoms, a concentration of the second chaotropic agent contained the alcohol contained in the second buffer is at least one in the second buffer is higher than or equal to 0 .5 mol/ L selected from branched or linear alkanols respec - 60 and lower than or equal to a maximum solubility of the tively having 1 to 5 carbon atoms, second chaotropic agent in the second buffer, and each agent of the first chaotropic agent contained in the a concentration of the second surfactant contained in the first buffer and the second chaotropic agent con second buffer is 3 to 15 volume % . tained in the second buffer includes at least one 7 . The method of recovering short - chain nucleic acids compound selected from the group consisting of a 65 according to claim 1 , wherein guanidine salt, sodium iodide , and sodium perchlo a concentration of the cation salt contained in the second rate , buffer is 10 to 200 mmol/ L . US 10 , 323 , 241 B2 25 26 8 . The method of recovering short - chain nucleic acids wherein according to claim 1 , wherein the step ( b ) further comprises : when the first buffer contains the alcohol, the alcohol a step (b ') of adding at least one proteolytic enzyme to the contained in the first buffer is at least one selected liquid components recovered in the step ( a ) , to carry out from branched or linear alkanols respectively having an enzymatic reaction , and 1 to 5 carbon atoms, a step (b " ) of the alcohol contained in the second buffer is at least one bringing the second mixture into contact with the selected from branched or linear alkanols respec second nucleic acid adsorbent to adsorb the short tively having 1 to 5 carbon atoms, chain nucleic acids having the length of 1000 bases each agent of the first chaotropic agent contained in the or less , the short -chain nucleic acids derived from In10 first buffer and the second chaotropic agent con the whole blood sample onto the second nucleic acid tained in the second buffer includes at least one adsorbent, and then compound selected from the group consisting of a separating and recovering the second nucleic acid guanidinium salt , sodium iodide , and sodium per adsorbent from being into contact with the second 16 chlorate , mixture , after the step ( b ') , each surfactant of the first surfactant contained in the wherein the at least one proteolytic enzyme includes at first buffer and the second surfactant contained in the least one selected from the group consisting of second buffer includes an ionic surfactant including lysozyme, proteinase , proteinase K , and cellulase . sodium dodecyl sulfate or at least one non - ionic 9 . The method of recovering short - chain nucleic acids 20 surfactant having a polyoxyethylene group , the at according to claim 1 , wherein the silicic acid - containing least one non - ionic surfactant selected from the material is silica , magnetic silica , glass , or silicon dioxide. group consisting of polyoxyethylene ( 10 ) octylphe 10 . The method of recovering short - chain nucleic acids nyl ether , polyoxyethylene (20 ) sorbitan monolau according to claim 1 , wherein the short - chain nucleic acids rate , polyoxyethylene (20 ) sorbitan monopalmitate , are circulating DNAs . 25 polyoxyethylene (20 ) sorbitan monostearate , poly 11 . A method of purifying short - chain nucleic acids from oxyethylene ( 20 ) sorbitan monooleate , polyoxyeth a whole blood sample , comprising : ylene ( 9 ) octylphenyl ether, polyoxyethylene (23 ) a step ( a ) of lauryl ether , polyoxyethylene ( 20 ) cetyl ether , digi bringing a firstmixture into contact with a first nucleic tonin , and saponin , and each absorbent of the first nucleic acid adsorbent and acid adsorbent, the first mixture having an alcohol 30 the second nucleic acid adsorbent includes at least concentration of 0 to 5 volume % , the first mixture one selected from the group consisting of a silicic being prepared by mixing a whole blood sample with acid -containing material, zeolite , aluminum oxide , a first buffer containing at least one selected from a titanium dioxide , zirconium dioxide , kaolin , mag first chaotropic agent and a first surfactant, the first as netic particles , ceramics , and polystyrene . nucleic acid adsorbent to adsorb nucleic acids 12 . A kit for recovering short - chain nucleic acids com derived from the whole blood sample onto the first prising : nucleic acid adsorbent, and then a first buffer containing guanidine hydrochloride as a first separating and removing the first nucleic acid adsorbent chaotropic agent and a first surfactant, where from being in contact with the first mixture , to 40 a concentration of the guanidine hydrochloride is recover liquid components of the first mixture ; higher than or equal to 1 mol/ L and lower than or a step ( b ) of equal to a maximum solubility of the guanidine bringing a second mixture into contact with a second hydrochloride in the first buffer and nucleic acid adsorbent , the second mixture having an a concentration of the first surfactant in the first buffer alcohol concentration of 10 volume % or higher , the 45 is 1 to 10 volume % , the first buffer to be mixed with second mixture being prepared by mixing the liquid a whole blood sample to have an alcohol concentra components recovered in the step ( a ) with a second tion of 5 volume % or lower ; buffer containing an alcohol, a cationic salt , and at a first nucleic acid adsorbent to least one selected from a second chaotropic agent be in contact with the first buffer mixed with the whole and a second surfactant, the second nucleic acid 30 blood sample , to adsorb nucleic acids derived from adsorbent to adsorb short - chain nucleic acids having the whole blood sample onto the first nucleic acid adsorbent and a length of 1000 bases or less , the short - chain nucleic be separated and removed from being in contact with acids derived from the whole blood sample onto the the first buffer mixed with the whole blood sample , second nucleic acid adsorbent, and then so as to recover liquid components of the first buffer separating and recovering the second nucleic acid mixed with the whole blood sample ; adsorbent from being in contact with the second a second buffer containing an alcohol being isopropanol mixture including the recovered liquid components ; or ethanol, a second chaotropic agent including guani and dine thiocyanate , a second surfactant , and a cation salt , a step ( c ) of 60 the second buffer to be mixed with the liquid compo eluting the short -chain nucleic acids absorbed in the nents, to have an alcohol concentration of the second second nucleic acid adsorbent by bringing water into buffer being 10 to 60 volume % or higher ; contact with the second nucleic acid adsorbent and recovered in the step ( b ) and recovering the short a second nucleic acid adsorbent to adsorb short - chain chain nucleic acids from the second nucleic acid 65 nucleic acids having a length of 1000 bases or less, adsorbent, thereby purifying the short- chain nucleic from the second buffer mixed with the liquid compo acids from the whole blood sample , nents , US 10 , 323 , 241 B2 27 28 wherein a concentration of the second chaotropic agent in the second buffer is higher than or equal to 0 . 5 mol/ L and lower than or equal to a maximum solubility of the second chaotropic agent in the second buffer, a concentration of the second surfactant in the second buffer is 3 to 15 volume % , a concentration of the cation salt in the second buffer is 10 to 200 mmol/ L , each surfactant of the first surfactant contained in the 10 first buffer and the second surfactant contained in the second buffer includes an ionic surfactant including sodium dodecyl sulfate or at least one non - ionic surfactant having a polyoxyethylene group , the at least one non - ionic surfactant selected from the 15 group consisting of polyoxyethylene ( 10 ) octylphe nyl ether, polyoxyethylene (20 ) sorbitan monolau rate , polyoxyethylene ( 20 ) sorbitan monopalmitate , polyoxyethylene ( 20 ) sorbitan monostearate, poly oxyethylene ( 20 ) sorbitan monooleate , polyoxyeth - 20 ylene ( 9 ) octylphenyl ether, polyoxyethylene ( 23 ) lauryl ether, polyoxyethylene ( 20 ) cetyl ether , digi tonin , and saponin , and each absorbent of the first nucleic acid adsorbent and the second nucleic acid adsorbent includes at least 25 one selected from the group consisting of a silicic acid - containing material , zeolite , aluminum oxide , titanium dioxide , zirconium dioxide , kaolin , mag netic particles, ceramics , and polystyrene . * * * * * 30