JPS6061594A - Fixed rna - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for immobilizing rods on a solid support, such as cancer, Mediterranean anemia, hemophilia, bone disease (J'JH disorders, various genetic disorders, and other diseases). For basic research in expression detection, cell analysis, gene cloning and molecular culture technology;') l-471 Message (14
Regarding A.

According to one aspect of the invention, the meso- ge R1,+
8 porous V1 solid support J2tc fixed 1hi) 1℃
・Mesage HiJ A for applications such as disease detection, molecules/:I:A-technology, etc.
I'll give it to you.

Preferably, the solid support
A sweats from nitro soil U-su, na fron, glass fiber 4 and other <A Kyou 1 1. +-, It's a bastard.

The present invention also provides for forming a solution of whole cells or partial cells containing the message HNA in a photocopy field, and adding this solution while attaching the message HNA to a solid support. through a suitable solid support filter,
and multiplication 1jlE +,! In a preferred aspect, the present invention comprises a method for placing an immobilized message II NA on a solid support characterized by removing the non-message from the support. zl) Cells are treated with 1j1 inhibitor of protein synthesis and Ribonuc I/
Rinse in Aze 1 wash and remove.
Freeze-thaw: From Unakata'li tc, 4] 11 cells are lysed and proteins are dissolved into proteinaceous enzymes and thorium is used to iodide cell components (Yoshi, C) during covation. In Nato's Chaotropic J edition, 1 water and 1 cow solution dissolves 51.

d) Selectively pass the extracted mesogen RNA through a filter.

e) stabilize the old 4A-protein binding on the filter;
and cleaning with a solution that removes unwanted contaminants, etc.
and r) Leap into this RNA filter molecular hybridization! j1. −
1) A method characterized by incubating basic proteins and other molecules in a solution for acetylation.

Cell washing should be performed as described in 91 below.

Laboratory specimens of solid tissues should be subjected to at least two PBSC tests (fl
, 5M 1 phrase CI , ], OIIIM MyCJy
4.0.14M phosphate buffer, pl+6.8.2S
ttg/ml to nr+#--n-g L") f,
) 75 -1111 i S 4--++-f'+
1-' Single cell suspensions can be broken down into cell clumps by blowing/driving at low speed for 10-30 seconds in a 4N apparatus. Other tissue division methods that do not cause cell lysis may also be used. Cells are pelleted by centrifugation of dividing cells consisting of single cells or specimens (samples of blood, urine, sputum, lymph, etc.) or laboratory cultures of cells in PH8C for 10 minutes at 1000 x g. . Cells can be resuspended in cold PBSC and re-pelleted. The choice of resuspension buffer is governed by the desire to protect the internal RNA from degradation by "freezing" the protein synthesis machinery using cyclohexamide and low temperatures, such as potent bonuclease inhibitors. is used. Isolation of specific cell types by fractional centrifugation, density gradient centrifugation or other methods may be performed at this stage. Various Jj methods can be used for cell washing before microlysis. In the simplest case, cyclohexamide is added to the sample and the cells are then lysed.

Individual cells can be obtained from solid tissue, and certain cell types can be isolated from residual tissue11. There must be.

The cell lysis and protein removal method is preferably carried out as follows. Wash cells with 1 ml of 20 mM vanadyl ribonucleoside or other appropriate bonuclease inhibitor and 20μ,! Add 97me DNAasel. Cells were incubated at 37 °C for 20 min and ioO
Add μjj/ml of protease or other suitable protease. The suspension is freeze-thawed twice in a cold bath, such as a methanol-dry ice bath, to lyse the cells. This mixture is then held at a temperature typically 37° CVc to allow proteolysis;
RNA is exposed by 1) ribonucleases (by dividing the cells under conditions that do not degrade the NA, and 2) by removing proteins that [bind to or mask the RNA]. Furthermore,
The authors believe that certain types of protein removal during cell lysis are carried out by filtration and R.
It has been found that it helps bind NA to the filter.

Solubilization of cellular components using saturated NaI is preferably performed as follows. First, 250 g of solid NaI was
By adding QOm(! to hot water, supersaturated Na
A solution is made. NaI crystallizes out of solution at room temperature, but the l-axis can be redissolved by heating the mixture at 75° VC. Then 0.81
: 3m supersaturated NaJ heated to 6075° l II+
(!0) Add lysed protease-treated cells.

The final concentration of NaI is 100% saturated at 25°. Any concentration greater than 80 at 25° can be used satisfactorily.

Concentrations below 80 parts at 25° can optionally be used, but binding and retention of ltNA to the membrane (see below, 11(1)) is suboptimal.

Filtration of lysed fal+ cells is preferably carried out as follows. The Nal solution is passed slowly through the filter under moderate vacuum]-0 This solution can also be extruded under pressure, drawn through centrifugal force, or subjected to one gravity iC, unless the flow rate precludes binding of the RNA to the filter. It is possible to force it out. Nitrocellulose and glass fibers have been found to be satisfactory filter materials. Cellulose acetate is unsatisfactory because it binds little or no RNA.

RNA filter washing) T is feminine and G soil? It is done in a absent manner. Most of the cell debris passes through the filter, whereas 11 NA and certain DNA and other molecules bind to the filter material. After the Nal solution has passed, the filter is washed with distilled water. This wash tends to remove 1) l=lΔ ribosomal RNA,) transfer RNA and other undesirable contaminants, and helps immobilize the message RNA to the filter. Acids and alcohols can also be used instead of distilled water.

Acetylation of RNA filters is preferably performed as follows. R14A filter was newly prepared with 0.2 M + reethanolamine and 0.25
% acetaldehyde for 10 minutes at 25°.

This step acetylates basic proteins and prevents non-specific adhesion of the radioactive globe to the filter during single molecule hybridization. In addition to this step, filter-bound RNA
a S e is inactivated.

In addition, to improve the shelf life of the filters formed, they can be baked for 2 hours in 80'OVC to remove the water.

This immobilized mesage Hhl A filter can be processed (=J, for example, active injection cloning 1.) for molecular hybridization to the labeled probe, which is a N A molecule, and immobilized. 1 (+4 A fs□ January 4A used as a template, RN eight hei can be subjected to the enzymatic synthesis step of m white + i!4).

Any of a number of standardization techniques can be used for molecular hybridization to IINA or DNA probes. Detergent and protein filters.

F i C011, polyvinylpyrrolidone, poly(A)
(Jeffre
ys, A, ,T, and Fi, avel,]−,+
1. A.

Ce1112:429-/137.1977).

The most 1ii74-possible conditions for molecular hybridization of DNA probes to IINA filters are: l) N8-RN
A hybridization is preferential over DNA-DNA hybridization (because (VolyeLst6i, n, B, and Oi,
l 10r:, (, +if:4.

D,, BBRC75: 1 ] ] 27-1132.1
977), 70-90%il, Ilz mu-f mi)"
, 0.1 5-0.5'y self, Pl (6-8,: 7-45° and several hours. After hybridization, unreacted probes are hybridized with old JA filter and/or pre-1 + iti hybridization solution. The degree of hybridization of the probe (2) is a measure of the corresponding RNA sequence on the filter, and several methods including radioautography or scintillation. This can be accomplished by any method of the method. All pre-hybridization washing steps must be performed in a solution free of nuclease activity. The extent of hybridization of the probe to the message RNA filter must be determined by RN is proportional to the degree of gene expression in the cells obtained. For example, if the probe is a radioactive hemoglobin gene, a high proportion of radioactivity will be detected on the filter.
i's hemoglobin H1'4r isomer message It N
A indicates the presence of A, and tL also indicates that the cell in which RNA is produced actively expresses hemoglobin molecules.

For the synthesis of immobilized RNA, any of several conditions of reverse transcription can be used. Typically, (Efstra also j, adj, s, A, and V
jl, 1a-Komaroff, Gen++Lf
1611+-, jump, +ゝri1 ke, 2:1.5-3
3.1 97 ri), we &:';-1, <N8 twisters 100 lie ノI OOn+M L+
1+;, PH8,: U, 10mM, 1μg of oligo(
10 mV dithiothreitol, 1 m+4 deogycinucleoside triposphate ('1 piece z,
&C), (50111M KC) and incubated in 1 unit of reverse transcriptase l-''□4-4
). Filter in this solution: 37°'-C6
time, during which 1c II N A
A DNA complement of fijj +ll is formed, and this DNA complement arrives at the filter via the FINA template and remains at 2t. This CDNA-R11A filter-- several times 30 lnM tris-11cJ! ,
pH 7, 5, 41 nM MgC- and 0.5 mM 2-
Wash in Merkaf Toetahar.

The second DNA strand (,1, the next (child) synthesized with the first
,ru(Humphrjesetal,,I+uc,AC
,■<ec:,5:905-924,l! +78
). Filter 50 μ4 3o lnM Lr], s
-nc, i<, 2-pl (soaked in 7,5, 10
Incubate at 0' for 5 minutes. Remove the filter and cool the solution to: 37°U'.
t)mM 1. ris −+lC,'ノ, pH 7,5
, 8mM MgCl2, n1J2-mercazoto-1-tanol, 2mM deoxynucleoside I lyphosphate and 5 units of T:,c:o'] IN J8 polymerase 1, Frag III (l [l L8 Added.

The solution is incubated at 22° for 5 hours, extracted with phenol, extensively CC dialyzed against water, and lyophilized.

The precipitate was treated with 100 mM NaCj! to cleave the U-shaped hairpin and form the plant end. , 50 mM zinc sulfate, P114.5.1 mM zinc sulfate and 5
Dissolve in 25 µe of a solution containing 1 µl of endonuclease and incubate for 2 hours at 1 µl. This solution is extracted with phenol, extensively dialyzed against water, and lyophilized.

To add oligo(dC)dil to double-stranded DNA, the precipitate was 5111M M9Cj', 2, h old 2-mercaptoethanol, 0.6 + nM dO', L°1
1 solution in 100 μe of a solution containing P117.1 and 12.5 mM Hopes-Na0F (buffer, P117.
1-ru. After adding 100 units of terminal transferase and incubating for 5 minutes at 37°, the reaction is extracted with phenol, dialyzed extensively against water, lyophilized and stored at -20'.

This immobilized message RNA has a double-stranded D
The preparation of NA copies is then cloned into a vector with linearized, oligo(+,R:)-,-dil (Humi, +bries eL r+J,, Vu
c, Ac, Res, 5: 905-92/l,
1978).

Any of several conditions for protein synthesis on immobilized RNA can be used. Typically (P+
iJ11am and Jackson.

Eur, J. Biochem 67: 247-2
56.1976), RNA filter 5011e
(1) Wheat germ extract%30 +1+MKIJ, 0.8
mM spermidine, 11]M dithreitol, lmM
adenone triphosphate), 0.1 mM guanone triphosphate, unlabeled amino acids and 5 μβ
Incubate for 60 minutes at 30° in 100 μl of a solution containing 58 methionine (106CLII11).

Techniques for cloning characteristic genes are limited by methods of screening for recombinant clones of the gene of interest.

If nucleic acid probes are available for the screening method, (i) million clones can be screened.If nucleic acid globes are not available, one million clones can be screened.
Only 0.00 clones can be screened, and sometimes the screening fails altogether. By using the present invention, new cloning procedures can be developed in which screening for rin r,j - is separate from the requirement of nucleic acid probes. Furthermore, the new method substantially improves the method of forming initial clone banks.

The present invention will be further explained below with reference to Examples.

EXAMPLE 1 Binding of RNA to Glass Filters and Nitrocell +1-S Membranes The following experiments were performed to demonstrate the ability of the method to bind message IIN to filter materials.

Human) (e1-a) cells grown in tissue culture were labeled with radioactive uridine.

Radioactive RNA was purified from these cells by conventional phenol extraction. NaCJ purified radioactive 1 raw RNA
, with a poly(A) tail of n N
A (most of the RNA), and the body
(A) No tail]'(NA(IJ bosomal RNA A
and transfer RNA).

The column matrix is oligo(d″1′)-cellulose glass, vc+lTi L, and poly(A
) Contains JRNA 0. OIMt+, P[19 was eluted from the glass. Poly(Δ)-minus 11 H8 is 0.5MNfl (oligo(dT' in J)
) - twice through cellulose, in each case adsorbed I
I chose INA. Poly) containing RNA is NZ,
0.5 M in lC, bound to oligo(d'r)-cellulose, 0.01 M trjs, pH 9
Shim elution in:.

Over 80% of BoIJ(A')-containing RNA bound to the third oligo(,rr)-cellulose casino, whereas less than 1% of BoIJ(A)-minus RNA bound. there were.

Poly(A)-containing] 1st-IA and poly(A)-minus RNA A was removed from ethanol for 41 minutes,
;1 Convenience/[Buffer e.g. fl l, l-11,
+]-+t, IJI 7.0 Shikoryo 19 completed. One ant (1-1) is held at 37°.
60 molecules of ink were printed. These RNA solutions were each dissolved in 0.833 volumes at 75 °C.
f supersaturated Na1l (2,5: soyjn(!■120
) Which system and combination 1, 2. The color of the RNA was filtered using a glass fiber filter (Wha, tman (IFC) or (1, nitrocellulose membrane (5cblθj c: b・) ■ and!
Xt:IILN・It, l-1△85).

This first thing. These filters were then filtered with various solutions by suction.
C-1-8 Table 1 Value - "+1 radiant mortality associated with filter.

* - Measured for rapid cooling - [L's.

l 8X S SC = 2.7MN, tC-1', 0
．． 27 Mrif-'/acidsu1・riu),,P
ll 70 poly(A)-minus 104A is bonded to glass in Nal, but almost bal 8X u
<' = C (1,3M NaCf, 0.135 N + citric acid, Pll7) was removed by washing; nearly complete removal was achieved with distilled water.

The binding of nitrocellulose l\ of Bo II (/1)-minus RNA A is at least 1. To take advantage of this, poly(
△)--containing '4' RNA is dissolved in glass or nitrocellulose VC in Nal.
, Conclusion 'B' is useful for some cleaning methods, especially distilled water.
It was S-axis stable.

Poly(A)-containing RNA A was present in condition T above and effectively sweated on the filter.
A) - Minus RNA It is clear that most of A is not Odono r)sa, jt). To the best of our knowledge, poly(A)-containing A1 rules 4/\ are exclusively mesosage RNAs, and most mesophages 1A also include B11(A) tails, so this example (The method performed in ``C'' can be applied to most or all of the message RNA glasses or (nitrohirulose filters). We conclude that ) can be 710) (poly(Δ) of Φ − minus RN
To the extent that the A Message HN A and to the extent that certain Bo1. Sage RN8 is the condition we have developed with seven filters (combined by A-4
- Leaving one possible cow as a li4 and one small thing, 11 we G earth this ji4' rj-j i'i- is included in this patent: I6.

We have observed that similar results can be achieved using Kl, N+, +Cf04 or other stimulant tropic salts, and we include them in the method of the present invention. guess with something). Satisfied with a certain purpose1, the result may also be different.
Achieved using 1
1) This change is included in the method of the present invention. Finally, the method of the present invention includes filtering from irrational If;σ to 11
Any cleaning operation that does not remove the 8.8 and unreasonably interfere with subsequent processing steps may be included.

Example ■ In the same manner as in Example I, "from II e 1 a cells"
(A) RNA was immobilized on Nitrocellulose. RNA filter and then each J! The cells were incubated 4 times under li conditions, and the retention rate of 1 IA was measured.

RNA filters were incubated for molecular hybridization (Jeffreys', Δ1, J, and I''J,
il V L! ], ,l, .

R, A, Ce11.12:429-4:39, J! 17
7).

RNA filter with 0.45 M NaC, i', 0
．． 0715M, NaClt and 20 mM vanadyl phosphonucleoside at 65° for 30 min.
filtered, then 0.2% Ficoll, 0
．． Prewashed for 3 hours at 65° in the same solution containing 2% borivylpyrrolidone, 0.24 live blood 7111 albumin, and then injected with 50 μjJ/ml low molecular weight salmon sperm DNA, 10 ttg/me poly(,A )
and 0.1 tidodecyl sulfate) for 1 hour at 65° in a second solution containing IJ.

Prewash RNA filters for molecular hybridization at 25 p.
9/m132p DNA probe and incubated for 20 hours at 65°.

After hybridization, T(NA) filters were post-washed for 6 times for 5 min, each wash was washed for 65 min using a third solution that did not contain Bo'J(A).
I went at °.

More than 60% fixed ratio RNA remained for this hybridization procedure.

RNA filter I) under the conditions of NA synthesis (see part 1 of the description of the preferred embodiment) at 37°.
There was no loss of immobilized RNA after incubation for 7. - (91% retention rate).

There was no loss of immobilized 11NA when the RNA filter was incubated under protein synthesis conditions (see Part 3 of the Preferred Embodiment) at -30° for 60 minutes.

Example 111 Utilization of filter-bound RNA for molecular hybridization Purified message n+vA (poly(A)-containing RN
We have shown that A) can be bound to the filter material (Example 1) and that Jt can be obtained under conditions normally used for molecular hybridization (Strain Example 2). Next, we found that at least some RNAs are available for molecular hybridization.
j] Plan an experiment to show what can be done (2).

RNA was purified from human leukemia leukocytes as follows. That is, white blood cells collected by leukaphoresis were collected at 25μjj.
Wash once with phosphate buffered saline containing 7.1 (L) cyclohexamide and once with 7.1 (L) LS11 buffer containing 25 μg/fnl of cyclohexamide.
R8B = ,0001MNaC,ll1.0025
MMgCJ, 25.0025M tri, s, pli
7. ! 'i), white blood cells at 0.05 M 1ris,
p)l 8.1% sodium dodecyl sulfate s 20
Dissolved in mM governoral uridine and 25 μg/me cyclohexamide.

Lysed cells Ct of 1.39 for each me VC: ;!
! :: O 4 is usually added at about 45°.

Smell the solution at 25 degrees and 100,00t)X, 17 at '7
The % RNA pellet was collected by centrifugation for an hour. II
NA was precipitated from ethanol three times, and then poly(Δ)-containing RNA and poly(A)-minus (Δ) were purified in the same manner as in Example 1.

Poly(A)-containing 1RNA and poly(A)-minus R
Na is o, s i : It is made into a solution at 75° of the vesicle and is supersaturated NaI (2,5j) Ni1l 4- I I7
+120). The resulting solution was filtered through a nitrocellulose membrane, washed with distilled water, baked at 80°, and washed with the premixed solution described in Example 2. The RNA filter was then washed with 50 thiformamide, 3×SSC(::!;C-(J,l 5
Mtl;+Cj! and 0.014 M sodium citrate, ptl 7 )1, Q 5 M tris
, pH 7, 2, 1% diethyl birocarbonate 1 and 5
000 cp+11 radiation 1/4: (321・)D N
Incubate 1- for 20 hours at 37° in the A probe.
did. These conditions are favorable for molecular hybridization.

This probe was derived from avian myeloblastosis virus.
reverse transcriptase, oligo(dT) primer, 32Pd
Compositions described in CTP and other required publications (Efstratiadis, A., and Vt1la
Published<0111411'Off.

Genetic Engineering 2:
15-3 a, iri7'l) to collect white blood cells! j (A,)-containing]' I (NA Aiura's DNA
Making a copy is 8 rounds from vC.

After hybridization, the filters were washed as described for the 4'11 wash in Example 2, and each filter was then evaluated for radioactivity by scintillation coefficient IQ pc , i:.

Table 3 Values are expressed in CpH1 and radioactivity from 5 measurements.

Table 3 shows that significant hybridization occurs only for filters containing RNA, and molecules on the filter associated with hybridization can be destroyed by incubating the RNA filter in a solution containing RNA aSe. Ru. Based on this and other examples, it can be concluded that T2 message RNA immobilized on nitrocellulose is readily available for molecular hybridization.

Presumably, the same hybridization results could be obtained by binding the RNA to nitrocellulose filters or other filters in other chaotropic salts such as those described in Example 1. It is believed that other sources and/or purified R l-I A may be effective as well (see, eg, Example 1).

Pre-hybridization wash solution, cross-section (washing solution, post-hybrid wash solution and cross-section test 1) and other formulations also destroy RNA.
It can be used for 'Lz pQ

Example 1v σ) Message RNA from cells for the preparation of molecular hybridization
In this situation, if it is possible to directly precipitate Rl. Take, d) Endo 1. c f
Since the decision is made without doing anything, it is clear that the decision is made without doing anything.
I think it is j, so I do 1. Message 1 page N A &'t
, 11:, J+11 selectively binds to the nitrocetrose filter in Nal, and one N+11 lyses cells with a thick f, C, so it is possible to extract II from the crude lysate.
This is true in the 9th example in which it seemed reasonable enough to find conditions for N A M4 binding, maximizing binding in a form that is acceptable for molecular hybridization. It clarifies some important principles.

Three different cell lines with different numbers of genes, dm, and therefore different amounts of dm messages ItNA were grown in the laboratory and then treated with 25μ97M of cyclohexamide.
Added up to e. Cells were released from the culture flask using 0.25% trypsin and PE:C (0.5M N
aCA, 0.14M phosphate buffer, pH 6,8, 25t
tg/ml cyclohexamide). 0 filled cells/. jJ to 0.5 ml of 0.5 M NaC, l
',, 10mM QqC, 12,10mM tri
s, pH 7.2, ifm? Shushi, 20mM
I made it.

Cells were incubated at 37° for 30 min, then 10
Freezing and thawing was carried out twice in a -70° bath in the presence of 0 μjl/me of profiinase. Incubate lysed cells with proteinase at 37° for 30 minutes! ·did.

This partially deproteinized 11 cell lysate was dissolved in VC70° with 0.813 volumes of supersaturated Na1(2,fiji
N817mlH20) was added. This solution and its dilution in saturated NaI were passed slowly through a nitrocellulose membrane and the RNA filter was then washed with distilled water. A freshly prepared 0.2K4 filter containing 0.25% acetaldehyde was added to this filter (1.IA filter).
) Acetylate basic proteins, including RNA AaSe, by incubating for 10 minutes at 25° in +7 ethanolamine, then filtering the RNA filter at 800°C.
It was dried for 2 hours.

These filters were washed for molecular hybridization preparation as in Example 2 and hybridized to IL, ]+u DNA globes using the conditions detailed in Example 2.

After hybridization, the filters were washed to remove untreated gloves (Example 2) and then analyzed by scintillation word count.

The results are shown in Table 4.

Table 4 Values are the radioactivity expressed as C11111 and I from the 23x sample. Control cells have 1 copy of the 6m gene, Co1.0320 has 75 copies of the c3m gene, and Co1.0321 has 325 copies of the (1-) gene. proportional to the number of

From Table 4, the hybridization value is the intracellular (1111 message RN
1'1 is a reasonable function of A. Based on this experiment and a number of its flk experiments, the message l'lNA can be deposited from cells onto iG-contacted VC nitrocellulose, and this message RN
An appropriate part, perhaps all, of the ``if'' is used for molecular hybridization.
It can be concluded that it is possible. We have shown that RNA from any cell can be immobilized in such a way that molecular crosslinking
I fully expect this to be used. Modification of V, f, l/i is necessary to adapt the method to a particular cell type. For example, some cells have a high proportion of bonucleases that degrade message RNA when removed from their natural growth environment. Before cell division, cyclohexamide freezes protein synthesis,
Although l+NA degradation is minimized, in some cases VC requires stronger protein synthesis inhibitors. Furthermore, in some cases, the entire process step Vc;
Must contain NSE inhibitor M f+: Yes 11 [
Ru. Such inhibitors include sodium dodecyl sulfate, phenol, vanadyl ribonucleosides, diethylstilbamidine isothionate, and the like. The choice of inhibitor not only depends on the purpose of RNAase inhibition, but also includes the essential requirement of the method: lack of inhibition, i.e., the degradation of DNAascVc during the process.

From the above examples, the present invention provides a method for evaluating the amount of specific message RNA in a sample (1)
Therefore, it can be seen that there is an assay method for determining the expression of a specific gene in a test cell sample. In our experiments, message RNA was used as filter material II.
It was shown that the RNA poly(8) tail was attached to the RNA poly(8) tail. Therefore, the heteropolymer region of the message RNA is free to serve as a template in the synthesis of complementary DNA, complementary RNA, or protein. .

Agent Patent Attorney ± 71'lJ Akira NoContinued from page 10 Inventors Isadora Protosky0 Inventors David Gillispie, Pennsylvania, United States of America 1528 Flat Rock Road, Narbot, Pennsylvania, 19072, United States of America, Pennsylvania 19343 S Geren Moore, Maple Flower Road , Box 138 Procedure n
O Seinai (spontaneous) lft Sum 5 B (1"I C Mr. Commissioner of the Patent Office 1, Indication of Matter A'1 1981 Special y) Application No. I Ci3106 t
:j2, Name of the invention fixed RNΔ 3. Person making the amendment A11 What relationship Special J] Applicant's name: George Bresler (and 2 others) 4. Agent: 107 Address: Minato-ku, East 5A Akasaka 2'1lJ No. 22'1 No. 2
6 Mori Building 306 Bow Telephone 58, '3-r6, Target of correction

Claims (9)

[Claims] (1) Message RNA is immobilized on a porous solid support, which is one of the most useful methods for disease detection, molecular technology, etc. (2) The solid support is made of nitrocellulose, nylon, glass fiber or other material to which the message IN is attached; NA0 (3) Forming a solution of whole cells or partial cells containing message RNA in a cbaotropic salt and passing this solution through a suitable solid support filter while binding the message RNA to the solid support. A method for placing immobilized message RNA on a solid support, the method comprising passing through the porous solid support and removing non-message RNA from the porous solid support. (4) a) Wash the cells in an inhibitor of protein synthesis and an inhibitor of ribonuclease, and remove the nuclear DNA from DNA
a) lyse the cells by methods such as freeze-thaw and digest the 5 proteins upon incubation with proteolytic enzymes; c) lyse the cellular components with a chaotropic agent such as sodium iodide; c) d) filtering the extract through a filter that selectively binds message RNA; and e) filtering the extract through a filter that selectively binds message RNA. Wash with solution to remove, and f) hybridize the RNA filter with Leap! 4. The method according to claim 3, further comprising incubating basic proteins and other molecules that can be acetylated in a solution that acetylates them. (5) Kao I. Robitzk (C1] aO1, r01]]
, c) The method according to claim 4, wherein a supersaturated solution of sodium iodide is used as the j theory. (6) The method according to claim 5, wherein the supersaturated solution is at least 80 φ saturated at 25°C. (7) The solution is passed through the porous solid support under vacuum, centrifugal force or pressure.
The method described in Section 5, Section 5, or Section (). (8) Non-message RNA material is supported]-1V Wash the body with distilled water, acid or alcohol a) Extract 1. It is removed by! 1 Te 571 Claim No. 3 Nor, 1,
4Jl″i, 5th item, 5JJJ or the method described in 1th item 7. (9) The method according to any one of claims 3g4 to 8th J'fi of claim 5' for removing water by baking the solid support. (1(υ 'l'J'i'F Claims Items 3 to 9
1. A solid support for immobilized message RNA produced by the method of any one of Items 1 to 1. 0υ Claims 1 or 2), consisting of a solid support containing the immobilized message RIi A according to claim 0, ii) N A or RNA A 7' lobe, immobilized This glove is characterized by the fact that RNA is involved in molecular hybridization. (I2, claim 1 or 1) In the synthetic protein comprising a solid support containing immobilized message RNA A as described in o, +yi, the immobilized RNA A is synthesized by protein G or protein Q. A synthetic protein characterized by: