WO1996000228A1

WO1996000228A1 - Method for the rapid isolation of nucleic acid

Info

Publication number: WO1996000228A1
Application number: PCT/US1995/007940
Authority: WO
Inventors: Janice T. Brown
Original assignee: Dade International Inc
Current assignee: Dade International Inc
Priority date: 1994-06-23
Filing date: 1995-06-23
Publication date: 1996-01-04
Anticipated expiration: 1996-12-23
Also published as: AU2907995A

Abstract

A method is disclosed for isolation of DNA and substantially intact RNA from a biological sample. The invention comprises the steps of incubating the sample with a lysing buffer comprising an ionic detergent. The lysing buffer is substantially free of guanidine compounds. A salt solution is then added to the incubated mixture, and precipitated detergent-protein is separated from the nucleic acid in solution. A nucleic acid-precipitating agent is added to the nucleic acid solution, and the precipitated nucleic is recovered. Nucleic acid isolated according to the invention is suitable for cloning, construction of libraries and for use as a template in amplification reactions.

Description

Method for the rapi d i sol ati on of nocl ei c aci d .

Field of the Invention

This invention relates to isolation of nucleic acids from biological samples. More

particularly, this invention relates to methods and articles of manufacture useful for

isolating RNA or DNA from biological samples in a simple, rapid manner and to the use

of such isolated nucleic acid as a template in amplification reactions.

Background of the Invention

Procedures for the isolation of nucleic acids from biological samples are

well-known in the art. Isolated nucleic acids are useful for many biotechnology-related purposes such as gene cloning, analysis of translation products in vitro and detection of particular sequences for diagnostic assays. Isolated nucleic acids often serve as

templates for amplification protocols such as polymerase chain reaction (PCR),

self-sustained sequence replication (3SR) and nucleic acid sequence-based amplification.

Saiki et al., Science 239:487 (1988); Guatelli et al., Proc. Natl Acad. Sci. USA 87: 1874 (1990); U.S. Patent No. 5,130,238 to Malek, L., et al.

Some methods have been developed to isolate a particular type of nucleic acid

such as ribonucleic acid (RNA). One method for isolating RNA from biological samples

is described by Chirgwin, J.M. et al., Biochemistry 18:5294 (1979). This method yields

RNA that can be translated in an in vitro translation system and that is suitable for

synthesis of cDNA. However, the method requires several hours to complete and

involves several centrifugation steps. Another procedure for isolating RNA is described in U.S. Patent 4,843,155 to

Chomczynski, which procedure uses a composition comprising a guanidinium salt and

acid phenol. U.S. Patent 4,935,342 to Seligson and Shrawder describes a method for

isolating RNA by utilizing ion exchange columns. U.S. patent 5,155,018 to Gillespie

and Cuddy describes a method for isolating RNA using siliceous material, such as

finely-divided glass, in the presence of a concentrated, acidified chaotropic salt.

A particularly difficult aspect of RNA isolation is the presence of ribonucleases in

many biological tissues and specimens. Ribonuclease activity during the isolation of

RNA generally results in unwanted degradation of RNA species present in the sample. Ribonucleases are noted for their stability under conditions that denature most other

proteins, and for the ease with which denatured ribonucleases may renature. A major

concern in methods intended to isolate RNA is preventing ribonuclease activity

sufficiently to allow intact RNA to be recovered.

Many methods have been described for isolation of deoxyribonucleic acid (DNA)

from biological samples. One early procedure for isolating DNA from biological

samples is described in Marmur, J., J. Mol. Biol. 3:208 (1961). This procedure yields

DNA of relatively high purity, but is rather time-consuming. More rapid methods of

DNA isolation, such as known methods for rapid isolation of plasmid DNA from

bacteria, are described, for example, in Bimboim, H.C. and Doly, J., Nucl. Acids Res.

7: 1513 (1979) and in Holmes, D.S. and Quigley, M., Anal. Biochem. 114:193 (1981).

Other DNA isolation methods are disclosed in, for example, Vogelstein and Gillespie,

Proc. Natl. Acad. Sci. USA 76:614 (1979) and Gross-Bellard et al. Eur. J.

Biochemistry 36:32-38, (1973). Certain methods are intended to isolate total nucleic acids, including both DNA

and RNA. U.S. Patent 5,128,247 to Koller describes a method for isolating nucleic

acids from cells using a chaotropic agent such as guanidinium isothiocyanate and subsequent exposure to a polyanion-containing agent such as heparin. This method is

intended for isolation of high molecular weight nucleic acids for cloning. Another

method for isolating RNA and DNA is disclosed in U.S. Patent 5,010,183 to Macfarlane,

which describes a method using a cationic detergent.

Many of the known nucleic acid isolation methods suffer from one or more

drawbacks. For example, some methods require several hours or days to complete. Certain methods require column separation protocols that use expensive solid phase

supports. It is known that nucleic acids isolated by some methods are difficult to amplify

by PCR or 3SR. Thus, there is a continuing need for methods suitable for preparing

amplifiable nucleic acid templates. Further, there is a need for rapid, low cost methods

for isolating nucleic acids, for example, for use in a clinical assay. A clinical work-up of a biological sample in which nucleic acid will be analyzed generally requires that nucleic

acid be isolated, the assay protocol be performed, the results be interpreted and

analytical reports be prepared. A rapid nucleic acid isolation method would assist in the

completion of clinical assays in a short period of time, preferably within one day.

There also is a need for simple nucleic acid isolation methods that allow

processing of multiple clinical specimens. Many known isolation methods require

multiple steps and it would be desirable to have methods using fewer steps and having

fewer components. Summary of the Invention

A method is disclosed for isolating total nucleic acid from a biological sample

comprising RNA, DNA and protein, in which the isolated RNA is substantially intact.

The method comprises the steps of a) incubating the sample in a lysing buffer comprising

an ionic detergent, for a time from less than about 1 minute to about 120 minutes; b)

precipitating a major portion of the protein in the incubated sample by adding a salt

composition under conditions where the nucleic acid remains substantially in solution; c)

separating the precipitated protein from the nucleic acid in solution; d) precipitating the

nucleic acid from the nucleic acid solution using a nucleic acid-precipitating agent; and e)

recovering the precipitated nucleic acid. Notably, the lysing buffer is substantially free of

guanidine compounds such as guanidine thiocyanate.

The lysing buffer also may include a proteinase or a ribonuclease inhibitor. The

ribonuclease inhibitor may comprise vanadyl ribonucleoside complex.

The incubating step may occur for a time from less than about 1 minute to about

5 minutes and at a temperature from about 22°C to about 65°C. The method may further

comprise the step of extracting residual protein from the nucleic acid solution after the

separating step, using a protein-extracting agent.

Nucleic acid recovered according to the invention may be suitable for use as a

template in an amplification reaction. For example, recovered RNA may be effective for

cDNA synthesis and amplification by 3SR and recovered DNA may be effective for

amplification by PCR.

An article of manufacture is disclosed, comprising packaging material and a

lysing buffer within the packaging material. The lysing buffer comprises an ionic detergent, and is substantially free of guanidine compounds. The article further comprises a label or package insert accompanying the packaging material, which

indicates that the lysing buffer is suitable for isolating total nucleic acid according to a

method of the invention. An article of manufacture may further comprise a salt

composition, in which case the label or package insert further indicates that the salt

composition can be used to isolate total nucleic acid according to a method of the

invention.

Brief Description of the

Figure 1 is a photograph of an ethidium bromide-stained agarose gel showing the yield of nucleic acid isolated according to the invention.

Figure 2 is a photograph of an ethidium-bromide stained agarose gel. Lanes 1

and 6 are Lambda DNA digested with HinDIII. Lanes 2-5 show l/20th of the nucleic

acid isolated from various numbers of HeLa cells according to the invention. Lane 2, 1 x

10⁵ cells; lane 3, 5 x 10⁵ cells; lane 4, 1 x 10⁶ cells; lane 5, 5 x 10⁶ cells.

Figure 3 is an autoradiogram of a slot blot, showing 3SR reaction products prepared from template nucleic acid isolated in Figure 1. Slots A1-A3 show the products

prepared from nucleic acid in lanes 2-4 of Figure 1, respectively. Slots B1-B3 show the

products prepared from nucleic acid in lanes 5-7 of Figure 1, respectively. Slots C1-C3

show products prepared from nucleic acid in lanes 8-10 of Figure 1, respectively. Slot

Dl shows the product prepared from nucleic acid in lane 12 of Figure 1. Slot El shows

the product prepared from nucleic acid in lane 11 of Figure 1. Figure 4 is a photograph of an ethidium bromide-stained agarose gel showing the

yield of nucleic acid using the step of removing residual protein.

Figure 5 is a photograph of an ethidium bromide-stained agarose gel showing the

nucleic acid yield using different combinations of salt solutions and nucleic

acid-precipitating agents.

Figure 6 is an autoradiogram of a Northern blot of nucleic acid samples isolated

according to the invention, using a probe complementary to the bcr2-abl2. Lanes 1-3:

1, 5, and 10 μl, respectively, of nucleic acid isolated using 1.0 μg/ml proteinase K in the

lysing buffer; Lanes 4-6: 1, 5, and 10 μl of nucleic acid isolated without proteinase K in

the lysing buffer.

Figure 7 is a photograph of an ethidium bromide-stained agarose gel, showing

amplification of nucleic acid isolated according to the invention. Lane 1: 0X174

digested with Haelll; Lanes 2-4: 1, 5, and 10 μl, respectively, of nucleic acid isolated

from SiHa cells and amplified by PCR using primers MYl 1 and MY09; Lane 5: Control

reaction mixture using MYl 1 and MY09 primers without added template; Lanes 6-7: 2

and 7 μl, respectively, of nucleic acid from K562 cells converted to first strand cDNA

and amplified by PCR using primers BB164 and BB165; Lane 8: 7 μl of nucleic acid

from K562 cells amplified by PCR using primers BB164 and BB165 without conversion

to cDNA; Lanes 9-10: 2 and 7 μl respectively of nucleic acid from K562 cells converted

to first strand cDNA and amplified by PCR using primers BB 160 and BB 165; Lane 11 : 7 μl of nucleic acid from K562 cells amplified by PCR using primers BB160 and BB165

without conversion to cDNA; Lane 12: 7 μl of nucleic acid from K562 cells isolated by

RNAzol B, converted to first strand cDNA and amplified by PCR using primers BB164 and BB165; Lane 13: 7 ytl of nucleic acid from K562 cells isolated by RNAzol B,

converted to first strand cDNA and amplified by PCR using primers BB160 and BB165;

Lane 14: 0X174 digested with Haelll.

Figure 8 is a photograph of an ethidium-bromide stained agarose gel. Lanes 1

and 6 are 0X174 DNA digested with Haelll. Lanes 2-5 show different dilutions of the

PCR product from nucleic acid isolated from HeLa cells using the MY09/MY11 HPV

consensus primers. Lane 2, 1 :1000 dilution; lane 3, 1:750 dilution; lane 4, 1:500

dilution; lane 5, 1:250 dilution.

Figure 9 is a photograph of an ethidium bromide-stained agarose gel, showing

the PCR products of nucleic aid isolated from cervical swabs. The primers used in Row

1 were MY09/MY11 and the primers used in Row 2 were GH20/PCO4. Samples treated only with proteinase: lanes 2, 4, 6, 8 and 10 from samples 1, 2, 3, 4 and 5,

respectively. Samples treated according to the invention: lanes 3, 5, 7, 9 and 1 1 from

samples 1, 2, 3, 4 and 5, respectively.

Detailed Description of the Invention

The applicant has discovered methods that result in rapid isolation of total

nucleic acid from a biological sample, while utilizing few solution components and

utilizing inexpensive equipment. Methods of theinvention involve lysing a biological

sample in a lysing buffer, which solubilizes many cellular proteins and frees nuclear

proteins from DNA and RNA, followed by separation of nucleic acid from protein and

recovery of the isolated nucleic acid. Surprisingly, RNA present in the isolated nucleic

acid is substantially intact. Isolated DNA or RNA can be used as a template in an amplification reaction. In particular, isolated nucleic acid is useful as a template for 3SR or PCR. Further, RNA isolated by the method of the invention is suitable for cloning of

mRNA species. An advantage of the present invention is the rapidity with which nucleic

acid may be isolated from a biological sample. The method of the invention shortens

considerably the time required for completing an amplification-based diagnostic analysis

of a biological specimen, since the lysis of the biological sample may be completed in less

than about 1 minute.

A biological sample may comprise intact cells, clumps of cells, portions of cells, isolated nuclei, or tissue. A biological sample may be preserved, e.g., fixed, frozen

and/or embedded, provided that such preservation permits subsequent isolation of

substantially intact RNA and DNA. For example, a biological sample may be derived

from a specimen that has been preserved for archival purposes, e.g., fixed and embedded

in paraffin. A biological sample may be fixed and/or frozen to allow later analysis, e.g.,

at a laboratory distant from the clinic where the sample was taken or when a sufficient

number of samples have been accumulated for efficient isolation of nucleic acid.

Alternatively, a biological sample may be a fresh preparation, i.e, a specimen that is to

have nucleic acid isolated within a short period of time after the sample is taken, without

an intervening preservation step.

A biological sample may be from a source such as animal tissue or cells,

including without limitation a cell culture, intact tissue pieces (e.g., a biopsy), or a whole

organ. Alternatively, a biological sample may comprise plant or fungal tissue or cells, which may be processed before performing a method of the invention, for example, by

removing the cell walls. The amount of tissue or the number of cells present in a biological sample may be adjusted as desired, e.g., to achieve a desired yield of nucleic acid. For example, if cultured mammalian cells are the source of a biological sample and

the isolated nucleic acid is to be a template for an amplification reaction, a suitable

amount may be from about 500 to about 1 X 10⁷ cells.

A biological sample is incubated in a lysing buffer, which comprises an ionic

detergent. An ionic detergent such as sodium dodecyl sulfate (SDS) is thought to

solubilize and to quantitatively bind to proteins in the sample, allowing protein-detergent

complexes to be precipitated when the salt concentration of the mixture is increased in

the second step of the method. The concentration of ionic detergent in a lysing buffer is

generally from about 0.05% to about 5%, preferably from about 0.1% to about 2%,

more preferably from about 0.3% to about 1.5%.

Additional, optional components may be included in a lysing buffer if desired.

For example, lysing buffer may comprise an ionic detergent, a ribonuclease inhibitor, a

sulffiydryl reducing agent and a pH buffering agent. When isolation of substantially

intact DNA is desired, lysing buffer may comprise an ionic detergent and a pH buffering agent. However, additional components such as a sulffiydryl reducing agent and a

ribonuclease inhibitor do not interfere with isolation of DNA.

Ribonuclease inhibitors are optionally included in a lysing buffer to reduce

degradation of RNA in the biological sample during the isolation procedure. An

illustrative example of a ribonuclease inhibitor is vanadyl-ribonucleoside complex

(VRC). Vanadyl-ribonucleoside complex comprises a mixture of the complexes formed between an oxovanadium IV ion and each of the four ribonucleosides. These complexes

are transition-state analogs that bind to many RNases and nearly completely inhibit enzyme activity. Berger, S. and Birkenmeier, C, Biochemistry 18:5143 (1979). Other

suitableribonuclease inhibitors include malacoid and bentonite.

A sulffiydryl reducing agent optionally is included in the lysing buffer.

Illustrative examples of such reducing agents are dithiothreitol and β-mercaptoethanol.

A pH buffering agent is optionally included to ensure that no unwanted changes occur in

the pH of the lysing buffer. It is known that highly alkaline solutions can degrade RNA

and that highly acid solutions can precipitate and depurinate nucleic acids. Typical pH

buffering agents include Tris (hydroxymethyl)-aminomethane, sodium or potassium

phosphate and morpholino-propane sulfonic acid.

Lysing buffer optionally contains a proteinase, which may assist in freeing nuclear

proteins from the nucleic acids. Lysing buffer preferably contains a proteinase if a

biological sample comprises cell clumps or intact tissue pieces. Inclusion of a proteinase

is particularly preferred when the resulting nucleic acid is to be used as a template in an

amplification reaction. Illustrative examples of proteinases are proteinase K, Pronase™

(self digested), and pepsin. Such proteinases typically are present in a lysing buffer from

about 1 to about 500 μg/ml.

A lysing buffer according to the invention is substantially free of guanidine

compounds, which are potent irritants. Many previously known lysing buffers used in

RNA isolation have guanidine compounds as a component. For example, the lysing

buffers in U.S. Patent 4,843, 155 and the RNAgents® Total RNA Isolation System,

Catalog number Z5110, Promega Corporation, Madison Wl 53711-5399, have guanidine

thiocyanate as a component. As used herein, the term guanidine compounds refers to guanidine and guanidinium compounds and salts thereof, including, but not limited to

guanidine thiocyanate and guanidinium chloride.

The length of time allotted for incubation of a biological sample in lysing buffer

depends to some extent upon the nature of the biological material, e.g., species, cell

type, amount of sample and the like. Biological samples such as cultured mammalian

cells generally require from less than about 1 to about 120 minutes, preferably about 1 to

about 60 minutes, more preferably about 1 to about 30 minutes. Most biological

samples will require less than about 60 minutes. The amount of lysing buffer added to a

sample is sufficient to allow separation of protein and recovery of nucleic acids in

subsequent steps. The amount of lysing buffer is adjusted according to factors such as

species and tissue type of the sample, the amount of sample and the like. It will be

apparent to those skilled in the art that it may be useful to agitate the mixture before or during the incubation in order to facilitate disruption of the sample, e.g., shaking, vortexing, or equivalent agitation method.

A solution comprising a major salt and a minor salt is then added to the incubated

mixture. A major salt may be, for example, potassium acetate, potassium chloride,

sodium acetate or sodium chloride. A minor salt may be a salt such as magnesium

chloride or sodium chloride. When SDS is the ionic detergent, potassium and/or

magnesium ions are preferably used to form the salt composition since sodium salts are not as effective at precipitating SDS.

The amount of salt composition added to the incubated mixture is sufficient to

cause detergent-protein complexes to precipitate, e.g., in about 10X to about 100X

molar excess of the major salt compound to the ionic detergent in the lysing buffer. For example, when an equal volume of a salt composition is added to an incubated mixture

comprising 0.5% to 2.0% SDS, the concentration of the major salt is generally from

about 0.8 M to about 2.5 M, preferably from about 1.0 M to about 1.8 M. The

concentration of the minor salt is generally from about 25 niM to about 75 mM,

preferably from about 40 mM to about 60 mM.

After addition of the salt composition, precipitation of detergent-protein

complexes may be accelerated, if desired, by chilling the mixture on ice for a brief period

of time, typically about 1-5 minutes. It is not necessary in most cases to chill the mixture

for more than about 10 minutes.

Precipitated detergent-protein is separated from the supernatant, which contains

most of the nucleic acid, by any suitable means. For example, detergent-protein may be

separated by centrifugation of the mixture or by adding a protein-binding resin such as

Strataclean™ resin (Stratagene, La Jolla, California, 92037), Pro-Cipitate™ (Affinity Technology, Inc. New Brunswick, NY 08901) or other commercially available protein

removal matrix. Alternatively, detergent-protein may be separated by adding phenol and

centrifuging the mixture. If phenol is used in removing detergent-protein, it is preferable

to use phenol with a pH of about 7.5 or above when isolating total nucleic acids or a pH

of about 6.0 or below when preferentially isolating RNA, preferably a pH of about 4.5. After separation of the precipitated detergent-protein, the supernatant may

contain a residual amount of protein in addition to nucleic acids. It may be desirable for

some applications to remove residual protein, which can be accomplished using an

additional protein extraction step. Phenol is a known protein-extracting agent that is

suitable for removing residual protein, typically by addition of an equal volume of water-saturated phenol to the supernatant. Other protein-extracting agents may be used

if desired.

The use of phenol containing 8-hydroxyquinolone (e.g., at about 0.1%) is

preferred when ribonuclease inhibitors such as vanadyl-ribonucleoside complex are

present in lysing buffer, since 8-hydroxyquinolone will extract these inhibitors and

phenol will extract protein. Multiple extractions with phenol containing

8-hydroxyquinoline may be performed, if desired.

Total nucleic acid is precipitated from the supernatant (after extraction of

residual protein, if desired) by the addition of a nucleic acid-precipitating agent. Suitable

agents include 2 volumes of ethanol, 1/2 to 1 volume of isopropanol, or 1 volume of a

solution comprising 5% w/v cetyltrimethylammoniurn bromide (CTAB).

The precipitated nucleic acid is recovered by means appropriate to the end use of

the recovered nucleic acid. For example, nucleic acids may be recovered by centrifuging the mixture in order to pellet the nucleic acid precipitate. Alternatively, nucleic acids

may be recovered by filtration on nitrocellulose or glass fiber filters. Nucleic acids are

preferably recovered by centrifugation and the resulting pellet is then resuspended in an

aqueous solution.

The recovered nucleic acid is useful for a number of end uses. The nucleic acid

may be treated with RNase-free DNase and used to prepare cDNA libraries or to clone

specific mRNAs. The nucleic acid may be used to prepare genomic libraries or to clone

specific genes.

A particularly preferred use is as a template in an amplification protocol such as

3SR or PCR. It is known that clinical samples may be difficult to prepare for use in a nucleic acid amplification protocol. For example, an ongoing problem with diagnostic

assays for human papillomavirus (HPV) in cervical swab samples is the inability to

amplify target DNA by PCR. Since small amounts of clinical material may be available

for analysis, the amount of HPV-containing target DNA may be quite small. The present

invention overcomes such difficulties, since nucleic acid isolated from clinical samples

according to the invention is readily amplifiable.

The invention will be understood with reference to the following illustrative

embodiments, which are purely exemplary, and should not be taken as limiting the true

scope of the present invention as described in the claims.

EXAMPLE 1.

Isolation of Substantially Intact RNA and DNA

from K562 cells.

A sample of human K562 cultured cells was obtained from American Type

Culture Collection (Rockville, Maryland). K562 is a line of pleuroeffusion cells derived

from a 53 year old female suffering from chronic myelogenous leukemia (CML) in blast

crisis. The cells were grown in RPMI with 10% fetal bovine serum and maintained by

standard culture methods. MDCK cells (a canine kidney cell culture) were also obtained

from the American Type Culture Collection and were grown and maintained as

described for K562 cells.

Approximately 2 X 10⁶ MDCK cells and I X 10⁴ K562 cells were added as a

mixed preparation to each sample tube. Cells in each tube were mixed with 450 μl of

lysing buffer and the mixture incubated at 50°C. Lysing buffer contained 0.5% SDS, 0.1 M dithiothreitol and 50 mM Tris HCL, pH 7.4. Vanadyl-ribonucleoside complex and

proteinase K were included in the lysing buffer as indicated in Table 1. Lysing buffer was

stored at room temperature and was stable for about 14 days.

Table 1. Lysing Buffer Compositions Used to Isolate the Nucleic Acid Samples of Figure 1

Protein¬ Incubation

VRC ase K Time⁸

Lane^c (mM) (μ/MI) (minutes)

2 1 0.5 10

3 1 0.5 20

4 1 0.5 30

5 5 0.5 10

6 5 0.5 20

7 5 0.5 30

8 1 0 30

9 5 0 30

10 0 0.5 30

11 0 1.0 30

12 RNAzol B^b ^a Incubation time in the indicated lysing buffer. ^b RNAzol™ (Teltest, Friends ood, TX) was used to isolate nucleic acid according to manufacturer's directions ^c Corresponding lane in Figure 1

An equal volume of precipitating salt solution, containing 1.6 M potassium

acetate and 50 mM magnesium chloride, was added to the lysed sample. This mixture

was incubated for 5 minutes at 4°C, precipitating most of the protein in the sample. The

mixture was centrifuged at 4000 RPM for 5 minutes to separate the precipitated protein

from the nucleic acid in the supernatant. Nucleic acid in the supernatant was then

precipitated by the addition of an equal volume of isopropanol. The nucleic acid precipitate was collected by centrifugation. The nucleic acid pellet was recovered by resuspending in 50 μl of 10 mM Tris, 1 mM EDTA (TE), pH 7.4. A 5 μl aliquot of the

recovered nucleic acid was loaded on a 0.8% agarose gel which included 1 μl/ml

ethidium bromide and electrophoresed at 80 V for 1 hour. The results are shown in

Figure 1. Haelll-digested 0X174 and Hind Ill-digested λ DNA were used as molecular

weight markers (Lanes 1 and 13).

As shown in Figure 1, incubating the cells in lysing buffer for longer than about 10 minutes does not appear to significantly increase the amount of nucleic acid

recovered by the method of the invention. A visually observable amount of nucleic acid

was recovered in the absence of a proteinase in the lysing buffer, as illustrated in lanes 8

and 9.

EXAMPLE 2.

Isolation of Substantially Intact RNA and DNA

from HeLa cells.

HeLa cells were added to 400 μl of 0.5% SDS and an equal volume (400 μl) of

1.6M KC1, 50mM MgCl₂ was added to the sample immediately thereafter. The mixture

was placed on ice for 5 minutes, 800 μl of phenol:chloroform:isoamyl alcohol was added

and the mixture was immediately vortexed for 10 seconds. The resulting suspension was

centrifuged at 6,000 rpm for 5 min. The aqueous layer was collected and 800 μl of

isopropanol was mixed into the mixture. The precipitated nucleic acid was recovered by

centrifugation at 14,000 rpm for 15 min. The nucleic acid pellet was washed once with

70% ethanol and the pellet was resuspended in 50 μl TE, pH 7.4. Figure 2 shows l/20th of the resuspended nucleic acid after electrophoresis on a 0.8% agarose gel containing

ethidium bromide.

As illustrated in Figure 2, DNA and ribosomal RNA in the isolated nucleic acid

was intact and migrated as discrete bands.

EXAMPLE 3.

Amplification of Isolated RNA by 3SR.

This example teaches that RNA recovered in accordance with the invention can

be used as a template in 3SR amplification. A 5 μl aliquot of each nucleic acid sample of Example 1 was used as a template

in a 3SR amplification reaction in an RNase-free 1.5 ml Eppendorf tube. The reaction mixture contained 20 μl of a 5X buffer (containing 200 mM Tris HCI, pH 8.1,150 mM MgCl₂, 100 mM KCl, 50 mM dithiothreitol, 20 mM spermidine), 5 μl (15 pmol) of each

of the priming oligonucleotides, 20 μl of a 5X nucleoside triphosphate mix (35 mM

rNTP's, 5 mM dNTP's), and 45 μl of DEPC-treated H₂0. The sequences of the primers

used in the reaction (325 and 329) are shown in Table 2. Each mixture was heat

denatured at 65°C for 1 minute. Following denaturation, each tube was transferred to a 42°C water bath and incubated for 5 minutes. Thirty units of AMV reverse

transcriptase, 2 units of E. coli Ribonuclease H and 1000 units of T7 RNA polymerase

were added to each tube and the mixtures were incubated at 42°C for 60 minutes. Table 2. Oligonucleotides

SEQ. ID

No. Primer Nucleotide Sequence

1 325 AATTTAATAC GACTCACTAT AGGGAAGATG CTGACCAACT CGTGTGT

2 329 TGCAACGAAA AGGTTGGGGT

3 BB302 GCTGAAGGGC TTTTGAACTC TGCTTA

An aliquot of each reaction, representing 1/10 of the total volume, was denatured

in 90 μl of 7.4% formaldehyde and 10X SSC (Sambrook, J., et al., Molecular Cloning:

A Laboratory Manual, Vol. 2, 2nd edition, Cold Spring Harbor Laboratory Press, New

York, 1989) at 65°C in a water bath for 10 minutes. Aliquots were ice-chilled

immediately and loaded onto a nitrocellulose membrane through a slot-blot apparatus.

Nucleic acid in the aliquots was immobilized on the nitrocellulose by baking at 80°C.

The filters were prewetted with hybridization solution (6X SSC, 10X Denhardt's

solution, 10 mM Tris pH 7.4, 0.2 mg/ml sheared salmon sperm DNA and 1% SDS, then

hybridized at 55°C for 45 minutes with a ³²P -labeled oligonucleotide probe (SEQ. ID

No.: 3) that was complementary to the junction sequence of the bcr2-abl2 translocation

characteristic of chronic myelogenous leukemia in humans. Shtivelman, et al., Nature,

315: 550-554 (1985). After hybridization, the filters were washed three times at room

temperature for 5 minutes each using 1 ml buffer/cm² filter, 2X SSC, 0.1% SDS. The

filters were exposed to X-ray film at -70°C with one intensifying screen. The results of the 3SR amplification are shown in Figure 3.

As shown in the slot blot of Figure 3, nucleic acid isolated according to Example

1 was a suitable template for 3SR amplification. Those samples that had proteinase K included in the lysing buffer appeared to yield substantially more amplification reaction

product. The yield of amplified product was approximately the same at all incubation

times in lysing buffer with proteinase K.

EXAMPLE 4.

Removal of Residual Protein.

This example teaches the removal of residual protein that maybe present after

precipitation of the major portion of protein in the biological sample.

K562 cells and MDCK cells were collected and mixed in the proportions

described in Example 1. Nucleic acid was isolated as described in Example 1, except that after separating the precipitated protein, residual protein was removed from the nucleic

acid supernatant by various procedures. For one sample, 50 μl of Strataclean™ protein

removal resin (Stratagene, La Jolla, California) were added to the supernatant. The

mixture was vortexed and incubated for I minute and the resin was removed by centrifugation (Figure 4, lane 2). For three other samples, 450, 125 or 250 μl of Pro-Cipitate™ (Affinity Technology, Inc., New Brunswick, NJ 08901) were added to

the supernatant (Figure 4, lanes 3, 4 and 5, respectively) and the mixture was vortexed.

After incubating for 1 minute at room temperature, the supernatant was centrifuged to

remove the Strataclean™ matrix. After removal of the protein-extracting agent, nucleic

acids in the supernatant were precipitated with an equal volume of isopropanol and

resuspended in 50 μl of TE, pH 7.4. A 5 μl aliquot was loaded on an 0.8% agarose gel

and electrophoresed for 1 hour at 80 V. A Hind 111 digest of λ DNA was used as a size

marker (Figure 4, lane 1). As shown in Figure 4, the amount of nucleic acid recovered is

easily visible in an ethidium bromide-stained gel for all four samples. EXAMPLE 5.

Precipitation of Nucleic Acid

This example teaches that various salt compositions and nucleic

acid-precipitating agents may be used in accordance with the invention.

K562 cells and MDCK cells were collected and mixed in the proportions

described in Example 1. Samples were solubilized by vortexing at room temperature in 450 μl lysing buffer comprising of 0.5% SDS, 50 mM Tris pH 7.4, 0.1 M dithiothreitol,

and 5 mM vanadyl ribonucleoside complex. An equal volume of the salt solution

indicated in Table 3 was added to each sample and detergent-protein complexes

removed by centrifugation. The supernatant was precipitated with the nucleic

acid-precipitating agent indicated in Table 3. Protein was extracted from one sample

using Strataclean™ resin as described in Example 3.

The precipitated nucleic acid was resuspended in 50 μl of TE, pH 7.4, and a 5 μl

aliquot of each sample was electrophoresed in a 0.8% agarose gel containing 1 μg/ml of

ethidium bromide. Figure 5 shows a photograph of the gel; the lane numbers correspond

to those shown in Table 3. Lane 1 contains Hind ]ffl-digested λ DNA. The results

indicate that nucleic acid-precipitating agents such as isopropanol and CTAB are effective with either a KCl or a KOAc salt solution. Table 3. Nucleic Acid Isolation Solutions

^c KOAc = 1.6 M potassium acetate, 50 mM MgCl₂ used as precipitating salt solution. ^d - = No protein extracting agent used. ' Strataclean™ resin used as described in Example 3 to remove residual protein. ^f One-half volume (450 μl) isopropanol used as nucleic acid-precipitating agent. ^g Equal volume of 5% w/v CTAB, 50 mM Tris, pH 8.0,20 mM EDTA used as nucleic acid-precipitating agent. ^h Centrifugation for indicated RPM and time at 4°C to collect nucleic acid precipitate.

EXAMPLE 6. Demonstration of Substantially Intact RNA in Isolated Nucleic Acid. K562 cells contain the bcr2-abl2 and the bcr3-abl2 translocations of

chromosomes 22 and 9. Shtivelman, et al., Nature, 315:550-554 (1985). Nucleotide sequences for bcr and abl are in Genbank Accession Nos. 74469 and 81414,

respectively. The messenger RNA produced by the bcr-abl translocation is about 8

kilobases (kb) in length. A Northern blot was performed to determine the size of ^'bcr-abl

mRNA in nucleic acid isolated according to the invention. Total nucleic acid was

isolated from K562 suspension cells by solubilizing 1 X 10⁶ cells in 450 μl of lysing

buffer containing 0.5% SDS, 50 mM Tris pH 8.0, 0.1 M dithiothreitol, and 5 mM vanadyl ribonucleoside complex. An SDS-protein complex was formed by adding 450

μl of 1.6 M KCl, 50 mM MgCl₂ to the mixture, vortexing briefly and incubating on ice

for 5 minutes. Approximately 900 μl of Tris-saturated phenol containing 0.1%

8-hydroxyquinoline, pH 8.0, was added to the mixture. After vortexing, the mixture was

centrifuged for 10 minutes at 12,000 RPM and the supernatant was collected. Nucleic

acid was precipitated from the supernatant using an equal volume (900 μl) of

isopropanol and pelleted by centrifugation at 12,000 RPM for 10 minutes. The pellet

was resuspended in 20 μl TE, pH 7.4. One, 5, and 10 μl of the resuspended nucleic acid

was loaded onto a 1.0% formaldehyde gel and electrophoresed at 50V for 1.5 hours.

The gel was then soaked in DEPC-treated water for 45 minutes and in 20X SSC for 45

minutes and nucleic acids transferred by capillary transfer from the gel onto a

nitrocellulose membrane. After transfer, the nitrocellulose was prewetted with

hybridization solution (6X SSC, 10X Denhardt's solution, 10 mM Tris, pH 7.4, 0.2

mg/ml sheared salmon sperm DNA and 1.0 % SDS), hybridized with a ³²P-labeled

oligonucleotide (SEQ. ID No.: 3) complementary to the junction sequence of bcr2-abl2

and exposed to X-ray film. An autoradiogram of the blot (Figure 6) showed that the

probe hybridized to a band corresponding to the size expected for bcr-abl mRNA. This

result indicates that substantially intact mRNA was recovered from the isolated nucleic

acid. EXAMPLE 7.

Amplification of Isolated RNA by PCR.

This example teaches that RNA recovered in accordance with the invention can be used to generate a template suitable for PCR amplification.

Nucleic acid was isolated as described in Example 6 from K562 cells. The

precipitated nucleic acid was resuspended in 50 μl of TE, pH 7.4, and an aliquot was

used to synthesize first strand DNA. The reaction conditions used to synthesize first

strand cDNA were: 3 μl 0.1M dithiothreitol, 50 pmol random hexamer, 4.0 μl of dNTP (2.5 mM each nucleotide), 3.0 μl 5X buffer (250 mM Tris pH 8.3, 375 mM KCl, 15 mM MgCl₂), 1.0 μl RNAsin (40 units/μl), 1.0 μl superscript reverse transcriptase (200

units/μl BRL, Bethesda, MD), either 2 or 7 μl of resuspended nucleic acid and

diet.hylpyrocarbonate (DEPC)-treated water to a final volume of 30 μl. The reaction

was incubated for 30 minutes at 37°C.

One half of the first strand reaction mixture was used in PCR. Two pairs of PCR primers were used, BB 164/BB 165 and BB 160/BB 165. The PCR reaction contained 10

μl dNTP (2.5 mM each nucleotide), 10 μl 10X PCR buffer (lOOmM Tris pH 8.0, 500

mM KCl), 2.5 μl 50 mM MgCl₂, 50 pmol each priming oligonucleotide, and water to

bring the final volume to 99 μl. Each PCR mixture was incubated for 7 minutes at 95°C;

amplification was initiated by the addition of 1 μl of Taq DNA polymerase. The

thermocycle conditions were 35 cycles of 1 minute at 95°C, 1 minute at 55°C and 1

minute at 72°C. The final extension was completed by incubating the mixture for 7

minutes at 72°C. The PCR reaction products were electrophoresed on a 1.5 % agarose gel

containing 1 μl/ml of ethidium bromide at 100 V for 1 hour. A 489 bp fragment was the

expected product from primers BB164 and BB165. This fragment migrates on a 1.5%

gel at approximately at 600 bp, as is known in the art. Such a fragment is visible in

Figure 7, lanes 6-7. A 783 bp band corresponding to the reaction product expected from

primers BB160 and BB165 is clearly visible in Figure 7, lanes 9-10. For both primer

pairs, the reaction product from template nucleic acid isolated according to the invention

migrates at the same position as a positive control, as shown in lanes 12-13. No reaction

product is visible in control reactions in which RNA was not converted to cDNA, as

shown in lanes 8 and 11. These results indicate that RNA isolated according to the

invention is successfully converted to DNA suitable as a template for PCR.

Table 4. Oligonucleotides

Seq.

ΓJD

No. Primer Nucleotide Sequence

4 BB160 GACTGCAAAT GGTACATTCC G

5 BB164 TCTGACTATG AGCGTGCAGA G

6 BB165 ACTGCTCTCA CTCTCACGCA

7 MY09 CGTCCMARRG GAWACTGATC

8 MY11 GCMCAGGG C ATAAYAATGG

9 GH20 GAAGAGCCAA GGACAGGTAC

10 PC04 CAACTTCATC CACGTTCACC

EXAMPLE 8.

Amplification of Isolated DNA by PCR.

This example teaches that DNA recovered in accordance with the invention can

be used as a template in PCR amplification. Nucleic acid was isolated from SiHa cells as described in Example 6. The

precipitated nucleic acid was resuspended in 50 μl of TE, pH 7.4, and 1, 5, and 10 μl of

the nucleic acid was used as a template for PCR. The PCR reaction contained were 10

μl dNTP (2.5 mM each nucleotide), 10 μl 10X PCR buffer (100 mM Tris pH 8.0, 500

mM KCl,), 2.5 μl 50 mM MgCl₂, 50 pmol of each primer, water to a final volume of 99

μl. The PCR mixture was incubated for 7 minutes at 95°C and amplification initiated by

the addition of 1 μl of Taq DNA polymerase. The thermocycle conditions were the

same as the conditions used in Example 7. The primers used were MY09 and MYl 1.

MY09/MY11 amplifies a 449-458 base pair portion of the human papillomavirus (HPV)

LI coat gene of genital HPV strains. The PCR reaction product was electrophoresed at 100 V for 1 hour on a 1.5 % agarose gel containing 1 ytg/ml of ethidium bromide. As

shown in Figure 7, lanes 2-4, a 452 bp band was clearly visible. No reaction products

were visible in a control reaction that lacked a template (Figure 7, lane 5).

In a separate experiment, nucleic acid isolated from HeLa cells in Example 2 was

diluted and subjected to PCR as described above. Figure 8 is a photograph of a 1.5%

agarose/ethidium bromide gel of the PCR product. A fragment of the size expected for

this portion of the HPV LI coat gene is visible.

EXAMPLE 9.

Processing of Clinical Samples

Cervical swab samples were obtained from a referral clinic in Boston, MA.

Samples (100 μl) were incubated in 100 μl of 400 μg/ml proteinase K, 1% SDS, 25 mM

Tris-HCl, pH 8.5, and 0.5mM EDTA for 1 hour at 55°C. The proteinase K was denatured at 95°C for 10 minutes and a 10 μl aliquot of each sample was used as a

template for PCR. The primers used were MY09/MY11 and GH20/PCO4.

GH20/PCO4 amplifies a 296 bp region of the human β-globin gene and was used as a

positive control for the ability of the sample to serve as a template.

Out of fifteen abnormal clinical cervical samples, only two were weakly positive

by PCR for the presence of HPV. The amplification of ^β-globin by GH20/PC04 was successful in 9 out of the 15 samples, but should have exceeded 90% (14 out of 15

samples). Samples that cannot be amplified in a positive control reaction are considered

to be unsatisfactory for diagnostic assays that involve an amplification step.

An attempt was made to recover DNA in the processed samples by precipitating

one-half of the proteinase-digested mixture with ammonium acetate/ethanol. The

precipitated material was used as a template for PCR; the attempt was unsuccessful, in

that no additional samples could be amplified by the β-globin primers.

EXAMPLE 10.

Amplification of Nucleic Acid Isolated from Clinical Samples

An equal volume of 1.0% SDS was added to one-half (200 μl) of the digested

clinical samples of Example 9. Four hundred μl of 1.6 M KCl, 50 mM MgCl₂ was added

next and the mixture was set on ice for 5 min. An equal volume (800 μl) of

phenolxhloroforπv.isoamyl alcohol was added to each mixture and vortexed for 10

seconds. Each mixture was centrifuged at 6,000 rpm for 5 min. The aqueous layer was

collected and an equal volume (800 μl) of isopropanol was mixed into the material.

Nucleic acid was recovered by centrifugation at 14,000 rpm in a microfuge for 15 min. The visible pellet was allowed to dry at room temperature for one-half hour, and subsequently resuspended in 100 μl autoclaved distilled H₂0. A 10 μl aliquot of each

sample, as well as a 10 μl aliquot of same samples digested as described in Example 9,

was subjected to PCR. PCR was carried out as described in Example 7, using the

MY09/MY11 primer pair and the GH20/PC04 primer pair. Aliquots of the PCR

products were electrophoresed on a 2% agarose gel containing ethidium bromide for 45

minutes. The results for 5 of the samples are presented in Figure 9.

As shown in Figure 9, no products are visible in the proteinase-treated samples,

regardless of which primer set was used (Rows 1 and 2, lanes 2, 4, 6, 8 and 10). When

nucleic acid was isolated according to the invention, a ^β-globin PCR product was visible

in all samples (Figure 9, Row 2, lanes 3, 5, 7, 9 and 11). An I-IPV PCR product was

visible in three samples (Row 1, lane 3, 7 and 11). By isolating nucleic acid according to the invention, a significantly higher percentage of HPV-positive cervical specimens can be detected, compared to known isolation methods. The foregoing detailed description has been provided for a better understanding

only and no unnecessary limitation should be understood therefrom as some

modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Janice T. Brown

(ii) TITLE OF INVENTION: RAPID ISOLATION OF NUCLEIC ACID

(iii) NUMBER OF SEQUENCES:10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dade International Inc.

(B) STREET: 1717 Deerfield Rd. , Box 778

(C) CITY: Deerfield

(D) STATE: Illinois

(E) COUNTRY: USA

(F) ZIP: 60015-0778

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM PC Compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: MS Word for Windows 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Louise S. Pearson

(B) REGISTRATION NUMBER: 32,369

(C) REFERENCE/DOCKET NUMBER: BA1-4646

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 708/267-5373

(B) TELEFAX: 708/267-5376

(C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AATTTAATAC GACTCACTAT AGGGAAGATG CTGACCAACT CGTGTGT 47

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TGCAACGAAA AGGTTGGGGT 20

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Shtivelman, E. et al.

(B) TITLE:

(C) JOURNAL:Nature

(D) VOLUME: 315

(E) ISSUE:

(F) PAGES: 550-554

(G) DATE: 1985

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCTGAAGGGC TTTTGAACTC TGCTTA 26 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GACTGCAAAT GGTACATTCC G 21

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCTGACTATG AGCGTGCAGA G 21

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACTGCTCTCA CTCTCACGCA 20

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic (X) PUBLICATION INFORMATION:

(A) AUTHORS: Manos, M. Ting, Y. Wright, D. Lewis, Broker, . Wolinsky, S.

(B) TITLE:

(C) JOURNAL: Cancer Cei:

(D) VOLUME: 7

(E) ISSUE:

(F) PAGES: 209 -214

(G) DATE: 1989

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Durst, M.

Gissman, L. Ikenberg, H. zur Hausen, M.

(B) TITLE:

(C) JOURNAL: Proc. Natl. Acad. Sci. USA

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: 3812-3815

(G) DATE: 1983

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGTCCMARRG GAWACTGATC 20

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Manos, M. Ting, Y. Wright, D. Lewis, Broker, T. Wolinsky, S

(B) TITLE:

(C) JOURNAL: Cancer Cell

(D) VOLUME: 7

(E) ISSUE:

(F) PAGES: 209 -214

(G) DATE: 1989 (X) PUBLICATION INFORMATION:

(A) AUTHORS: Durst, M.

Gissman, L. Ikenberg, H. zur Hausen, M.

(B) TITLE:

(C) JOURNAL: Proc. Natl. Acad. Sci. USA

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: 3812-3815

(G) DATE: 1983

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GCMCAGGGWC ATAAYAATGG 20

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Saiki, R. , et al.

(B) TITLE:

(C) JOURNAL: Science

(D) VOLUME: 239

(E) ISSUE:

(F) PAGES: 487

(G) DATE: 1988

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GAAGAGCCAA GGACAGGTAC 20

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: synthetic (X) PUBLICATION INFORMATION:

(A) AUTHORS: Saiki, R. , et al.

(B) TITLE:

(C) JOURNAL: Science

(D) VOLUME: 239

(E) ISSUE:

(F) PAGES: 487

(G) DATE: 1988

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAACTTCATC CACGTTCACC 20

Claims

WHAT IS CLAIMED IS:

1. A method for isolating total nucleic acid having substantially intact RNA from a biological sample comprising RNA, DNA and protein, said method comprising the steps of: a) incubating said sample in a lysing buffer comprising an ionic detergent, said lysing buffer being substantially free of guanidine compounds, said incubating occurring for a time from less than about 1 minute to about 120 minutes; b) precipitating a major portion of said protein in said incubated sample by adding a salt composition under conditions wherein said nucleic acid remains substantially in a nucleic acid solution; c) separating said precipitated protein from said nucleic acid solution; d) precipitating said nucleic acid from said nucleic acid solution using a nucleic acid-precipitating agent; and e) recovering said precipitated nucleic acid.

2. The method of claim 1, wherein said lysing buffer further comprises a proteinase.

3. The method of claim 1, wherein said lysing buffer further comprises a ribonuclease inhibitor.

4. The method of claim 3, wherein said ribonuclease inhibitor comprises vanadyl ribonuclease complex.

5. The method of claim 3, wherein said incubating step occurs for a time from less than about 1 minute to about 5 minutes.

6. The method of claim 1, further comprising the step of extracting residual protein from said nucleic acid solution using a protein-extracting agent, said extracting step occurring after said separating step.

7. The method of claim 1, wherein said incubating step occurs at a temperature from about 22°C to about 65°C.

8. The method of claim 1, wherein said recovered nucleic acid is effective as a template in an amplification reaction.

9. The method of claim 8, wherein said recovered nucleic acid comprises RNA effective as a template for amplification by self-sustained sequence replication.

10. The method of claim 8, wherein said recovered nucleic acid comprises RNA effective as a template for amplification by nucleic acid sequence-hased amplification.

11. The method of claim 8, wherein said recovered nucleic acid comprises DNA effective as a template for amplification by polymerase chain reaction.

12. An article of manufacture, comprising:

a) packaging material;

b) a lysing buffer within said packaging material, said lysing buffer comprising an ionic detergent, said lysing buffer being substantially free of guanidine compounds; and

c) a label or package insert accompanying said packaging material, said label or package insert indicating that said lysing buffer is suitable for use in a method for isolating total nucleic acid having substantially intact RNA from a biological sample comprising RNA, DNA and protein, said method comprising the steps of:

i) incubating said sample in said lysing buffer for a time from less than about 1 minute to about 120 minutes; ii) precipitating a major portion of said protein in said incubated sample by adding a salt composition under conditions wherein said nucleic acid remains substantially in a nucleic acid solution;

iii) separating said precipitated protein from said nucleic acid solution;

iv) precipitating said nucleic acid from said nucleic acid solution using a nucleic acid-precipitating agent; and

v) recovering said precipitated nucleic acid.

13. An article of manufacture as recited in claim 12, further comprising a salt composition, said label or package insert further indicating said salt composition is suitable for use in said nucleic acid isolation method.