what's new: the role of chaotropic salts in two-phase gene diagnosis

272 BioEssays Vol. 1, No. 6

The Role of Chaotropic Salts in Two-Phase Gene Diagnosis David Gillespie, Mary Jo Caranfa and Joel Bresser

Sum ma ry

The preparation of samples for gene diagnosis is time-consuming, labor-inten- sive and costly. These problems can be overcome by dissolving a biological source in a strong solution of a chaotropic salt, thenjltering the solution through an immobilizing membrane. Conditions exist for causing the selective immobilization of either DNA or mRNA. The resultant membrane is immediately ready for reaction with labeled gene probes.

Types of Gene Diagnosis

Gene diagnosis is the determination of abnormal DNA quantity or structure in a biopsy specimen or the determination of abnormal mRNA quantity or structure in a biopsy specimen. In virtually all systems under exploration the deter- minations are made by molecular hybridization of immobilized DNA or mRNA with complementary nucleic acid probes. Undoubtedly, future gene diagnosis systems based on molecular hybridization will be performed in a one-phase (liquid) system. Further ad- vances in gene diagnosis systems will undoubtedly use nucleic acid sequencing. However, since one-phase hybridization requires a means of hybrid detection not yet available and since DNA sequencing procedures are neither economical enough nor convenient enough to adopt to a ‘screening’ mode, we focus here on two phase molecular hybridization systems.

Gene diagnosis systems must have certain features if they are to be more useful than other diagnostic tests (Table I). They must yield information not otherwise obtainable or they must be capable of rapid and economical execu- tion on a screening basis. To be competitive with monoclonal antibody assays, one individual must be able to perform over 100 gene diagnosis tests/ day for under $5 per test with a turn-around-time of 2 h. Additionally, a successful gene diagnosis test must be convenient, reliable, sensitive and yield results which are immediately interpre- table to clinicians.

Two Dhase gene diagnosis consists of

TABLE 1. Requirements of gene diagnosis

1. Reliability, sensitivity, high signal/noise, quantitative - like research 2. Speed - under 2 hours 3. Simplicity -must be performable by untrained laboratory technician and machine 4. Economy - under $5 per test 5. Interpretability - physicians must be able to understand and assimilate results without re-education

how to optimize steps 2 and 3. Molecular hybridization (step 2) must be carried out with a vast excess of single-stranded DNA or RNA probes, preferably with reagents which accelerate the hybridization rate. It is within the present technical capability to achieve ‘com- plete’ hybridization of probe in under 1 h. Hybrid formation can be so specific that a single altered nucleotide can be identified,’ or so ‘relaxed’ that any of a group of related organisms can be detected. ‘Hybrid detection’ (Step 3) can now be accomplished in a matter of minutes, using biotinylated probes2 or enzymatically labeled probe^.^ Though the technology for steps 2 and 3 is not yet optimal for gene diagnosis it is clear that it soon will be.

How to optimize steps 1 and 4 is not as obvious. Nucleic acid immobilization by filtration can aid in purification of nucleic acids from other cellular components (Fig. 1) and can separate hybridized probe from unreacted p r ~ b e . ~ , ~ Nucleic acids can be immobilized on porous membrane^,^ or in cells on slides6 We focus here on the membrane alternative, without mini- mizing the utility of in situ molecular hybridization.

Purified DNA and RNA can be immobilized on nitrocellulose membranes using solutions containing NaCl. Except in isolated instances, however, NaC1-based systems will not be useful

TABLE ZI. Gene diagnosis

for gene diagnosis because of the requirement to purify the relevant nucleic acid (Table 111, items 4-5). Southern transfers and Northern transfers require nucleic acid purification and fractionation, but they yield information on DNA and RNA structure, respec- tively. These popular research methods will probably not succeed as gene diagnosis techniques, however, because alternative procedures using special probes and special electrophoretic techniques will eliminate many of the present steps.

Immobilization for DNA Diagnosis

But how can nucleic acid puriJication be overcome? A system was needed wherein cells could be dissolved essentially into molecular components, then exposed to an immobilizing medium under conditions where only the molecular species of choice (mRNA or DNA) would become bound.

Grunstein and Hogness’ (see also Table 111, item 3), first illustrated this principle in a method called the ‘colony lift’. Bacteria were grown on nitrocellulose as colonies, then converted to protoplasts. The cells were partially dissolved by exposure to NaOH/NaCI, a treatment which also denatured bacterial DNA and degraded bacterial mRNA. The NaOH was partly neutra-

l . Sample preparation 2. Molecular hybridization 3. Hybrid detection 4. Data handling

Y

four steps (Table 11, Fig. 1). It is obvious The most ignored step is SAMPLE PREPARATION!

tissues tissues fluids fluids

heat

S + S N C

Positive Negative

Fig. 1. Gene diagnosis schematic. A biological sample is prepared in such a way as to expose DNA in a form which will be reactive with a gene probe. The example given in thefigure is to dissolve cell constituents with Nal exposing the DNA, heat the dissolved cells to denature the DNA, then to selectively immobilize the denatured DNA by filtering the resultant solution through a Schleicher and Schuell nitrocellulose membrane ( S & S NC). Molecular hybridization is then performed by incubating the DNA-containing membrane under hybridization conditions’6 with a labeled ‘probe’. Gene diagnosis probes in the short term will carry radioactive labels, but in the longer term will employ nonradioactive labels.z’

TABLE IIZ. Sample preparation for selective DNA immobilization

Procedure Time Reliability

1. Dissolve in chaotrope Under 10 min Reliable 30 min Reliable 2. Protease, dissolve in chaotrope

3. Dissolve in NaOH, wash NaOH/NaCl, Tris/NaCl 30 min Unreliable 4. Quick phenol extraction 2 h Fairly reliable (losses,

5. DNA extraction 2 days Fairly reliable (losses)

~

co-immobilizants)

BioEssays Vol. 1 , No. 6 273

WHAT’S NEW

1 2

lized, then the ‘colonies’ of lysed bacteria were sucked through the membrane. Many cellular constituents passed through the nitrocellulose, but much of the DNA stuck. After irrever- sibly immobilizing the DNA on the membranes by ‘baking’, they could be probed for specific sequences by molecular hybridization.

While this creative technique illustrated the principle of selective immobilization from complex mixtures, it has drawbacks which compromise its use in gene diagnosis (but see ref 8). The technique yields only semiquantitive immobilization of DNA. Coimmobili- zation of protein interferes with hybridization. The required baking step tends to aggravate the problem because coimmobilized molecules became ‘stickier’ and increase nonspecific background.

It became clear that new systems had to be developed for nucleic acid immobilization from whole cells. In 1979, Vogelstein noticed that NaI promoted

the selective absorption of double- or single-stranded DNA to glass powderg and in 1983 Bresser showed that NaI promoted the immobilization of single- stranded DNA on nitrocellulose and nylon membranes.l0 NaI is one of a group of salts classified by Hamaguchi and Geiduschek as ‘chaotropic’.l’ Among the properties of chaotropic salts are their ability to dissolve cellular structures and the ability to destabilize DNA. Taken together (Table IV), these properties probably accounted for the success in 1984 of NaI-promoted selective DNA immobilization directly from whole cells.12

The conditions for NaI-promoted DNA immobilization are compatible with the requirements for successful gene diagn0~is.l~ As indicated in Fig. 1 cells are briefly exposed to proteolytic enzymes, made about 12 molal in NaI, heated for 10-20 min, then filtered. Fig. 2 presents typical results probing for Hepatitis B virus (HbV) DNA sequences in mononuclear blood cells of

Fig. 2. Detection of HbVsequences in mononuclear bloodcellsof advancedcancerpatients. Mononuclear cells were purified from the blood of sixteen individuals and DNA was immobilizedfrom them on to nitrocellulose by the Nalprocedure (reJ 12, Fig. I and text). The entire procedure, from receipt of cells to immobilized DNA was carried out in under 1 h. The DNA-containing membrane was incubated with a azP-labeled HbV DNA probe under molecular hybridization conditions and hybrih were detected by radioautography.

patients. Sixteen samples were handled simultaneously. The elapsed time from receipt of the cells to having immobilized DNA ready for molecular hybridization wasunder 60 min. Sample 1 (top sample, row 1) was a positive control patient with acute myelogemous leukemia who was also Hbs-antigen-positive, indica- tive of active HbV multiplication. The sample below it (second sample, row 1) represented blood from a normal, uninfected individual. The remaining fourteen samples were from Hbs- antigen-negative acute leukemia patients. Of those, two were positive for HbV DNA sequences. Thus, as has been found for hepatoma patients.14 HbV


WHAT'S NEW

DNA can be detected in blood leuko- cytes in the absence of circulating viral antigens. Using the NaI technology blood cells can be handled in batches of 100 or more, in which case the cost per sample is well under $5.

The immobilization is selective for DNA for the following reasons. The immobilization of proteins is poor in NaI and is driven down to about 0.1 % of the input by the protease treatment. The protease step can be eliminated when under 0.3 mg of cells or tissue is used. RNA is destroyed during the heating step. Small molecules pass through the membrane. Whether poly- saccharides and lipids are immobilized is not known, except that fatty tissues have been processed successfully.

Other chaotropic salts will promote DNA immobilization. We studied the sodium salts of trifluoroacetate (TFA), trichloroacetate (TCA), isothiocyanate (SCN), perchlorate (PCA), iodide and bromide (Fig. 3). In the absence of detergents the binding of single-stranded DNA to nitrocellulose was promoted with saturated NAI, 5 M-NaI, 5 M-GuHCL, 5 M-NaSCN, 5 M-KBr, 5 M-KCI, 5 M-NaCl, 5 M-KAc, 6 M-NaTFA (not shown) and, to a lesser extent, with 5 ~ - P C A . Efficient DNA binding was obtained only at low temperature with 5 M-NaSCN and 5 M-KSCN. Little to no binding was obtained with 5 M-NaPCA, 5 M-NaTCA, 5 M-GuSCN or water. DNA binding to nylon was generally the same as to nitrocellulose except that it was promoted by 5 M-GuSCN and H,O.

The addition of Brij 58 markedly suppressed binding of single-stranded DNA to nitrocellulose, except in 5 M-NaI, 5 M-GuSCN and hot KC1. Additionally, the binding of DNA to nylon in 5 M-NaPCA was not suppressed by Brij 58.

Thus, while chaotropic salts usually promote DNA immobilization, excep- tions have been noted. Those that do promote efficient DNA immobilization show subtly different characteristics and requirements. Finally, the various chaotropic salts have different abilities to solubilize any given biological sample. Probably, the general concept of using chaotropic salts for sample preparation in gene diagnosis is a useful one, but specific solutions and conditions will have to be developed in each case.

Immobilization for RNA Diagnosis

mRNA can also be selectively immobilized in NaI. The conditions for NaI-

promoted mRNA immobilization are also compatible with the requirements for gene diagn0~is.l~ Cells are briefly exposed to proteolytic enzymes, then to detergents. They are made about 12 molal in NaI and filtered. Fig. 4 presents typical results probing for oncogene expression in mononuclear blood samples of cancer patients treated with interferon (IFN). Mononuclear cells were purified from blood of two CML patients and divided into two aliquots. mRNA was immobilized from concentrated cells of one aliquot and from 1 : 4 dilutions (top four dots of each row). DNA was immobilized from concentrated cells of the second aliquot and from 1 :4 dilutions (bottom 4 dots of each row). Immobilized mRNA and DNA were hybridized with a radioactive gene probe corresponding to the abl oncogene. Fig. 4 shows that IFN treatment reduced the expression of the abl oncogene in two patients studied. The increase in hybridization to DNA in the post-IFN samples resulted from a larger sample size, and not from gene amplification (data not shown).

This immobilization protocol is selective for RNA. The binding of DNA is suppressed by detergents. Furthermore, any double-stranded DNA which be- comes entrained in the membrane remains double-stranded throughout the hybridization process (since there is no baking step required) and does not participate in molecular hybridization.

Other chaotropic salts promote mRNA immobilization. Manser and GefterI5 showed that potassium isothiocyanate was useful for measuring mRNA levels in hybridoma cells.

Efficient mRNA binding in chaotropic salts is more difficult to achieve than efficient DNA binding. Efficient immobilization of purified mRNA from NaI is unusual and in our hands is achieved only with RNA prepared freshly from oligo dT cellulose. More routinely 10-20% immobilization of purified mRNA is obtained. Manser and GefterI5 report that purified mRNA will not bind nitrocellulose at all in potassium isothiocyanate.

The efficiency of immobilization of mRNA from cells is also variable. Judging from unpublished results of several investigators the mRNA immobilization efficiency within one set of experiments is uniform. However, widely different efficiences have been communicated to us by different investigators. The reason for this variation is not known but is clearly important to establish.

It is apparent that in the short term the major use of chaotropic salts in gene diagnosis will be in measuring DNA sequences. This is not simply because technical problems still remain in mRNA immobilization but ako because it is not yet clear how to handle data from mRNA diagnoses in a clinically meaningful way (see below).

S N N N G G P T K T a K a a K U U C C B F C A I I I T H A A r A I c

nitrocellulose

no detergent

NYTRAN

nitrocel Iu lose

with detergents

NYTRAN

Fig. 3. Binding of DNA to nitrocellulose andnylon in various chaotropic salts. Single-stranded DNA labeled with 32P was adakd to the indicoted chaotropic salt with or without 1% Brij 58 heated to 95 "C for 20 min, then firtered through various membranes, while stifl hot or after being allowed to cool lo room temperature. The cooling period was not long enough to permit DNA reannealing before filtration. DNA binding to various membranes was compared in severalsalt solutions. The results shown were obtained with BA85 nitrocellulose membranes and NYTRAN nylon membranes. Gene screen-plus, Genentran, Posidyne and Biodyne nylon gave essentially the same DNA binding results as NYTRAN. The topmost ofpaired rows of dots wasfiltered hot while the bottommost was allowed to cool to room temperature after heating. GUT is SM guanidinium isothiocyanate.

BioEssays Vol. 1, No. 6 275

WHAT'S NEW

TABLE IV. Advantages of NaI B A B A

1. Promotes nucleic acid denaturation 2. Promotes nucleic acid binding to membranes 3. Dissolves cell structures

The Data Handling Problem

Gene diagnoses which measure the quantity of a DNA sequence is finding its first usefulness in the area of infectious disease. In theory, this represents a plus-or-minus situation where an infecting protozoan, bacterial, viral, etc., genome is either present or absent. DNA from the biological specimen is immobilized on a membrane, then hybridized with a probe representing the genome or a select portion of the genome of the infecting organism under scrutiny. However, even this simple case is confounded by the gray area between positive and negative results.

Negative results like that schemati- cized in Fig. 1 are not truly zeros. The labeled probe has some finite opportu- nity to form imperfect molecular hybrids with 'unrelated' DNA sequences, as might be formed with the genome of the cells of the tissue being analyzed. To a large extent these imperfect hybrids can be minimized by using 'stringent' molecular hybridization conditions and hybrid detection conditions which allow for only the most stable hybrid molecules.16 The labeled probe can also interact with molecules which co- immobilize with the DNA through electrostatic and hydrophobic interactions and with the immobilizing membranes itself. These 'background' interactions can be minimized by using the most selective immobilization procedures and by choosing postwashing conditions which destabilize the un- wanted interactions (e.g. include washes with high salt concentrations and detergents).

A second problem in handling data even from these ' plus-or-minus' experiments is more medical than scientific in nature. The DNA technology can reveal positives which appear negative by other criteria. In some cases, gene diagnosis is simply more sensitive than the pre-existing methodology, as in the Salmonella test in foodstuffs developed by Integrated Genetics while in other cases infected individuals are simply missed by the pre-existing methods as in the case of covert HbV. In either instance, the DNA test yields new information which was not part of the previous medical repertoire and therapy

strategies or regulatory strategies need to be developed before such results become commonly used.

More quantitiative and therefore more complicated gene diagnosis assays present additional problems in data handling. The problems in quantitative measurements, apart from deciding what is medically 'positive' are that the biological interpretations of the result are often obscure and that the technology used often precludes the average physi- cian from understanding the basic nature of the results. It is our opinion that a requirement to reeducate physicians is a highly undesirable quality in a proposed gene diagnosis test and therefore that an important desirable feature is the presentation of results in a context that is easily assimilated by clinicians. This is the primary reason for our earlier statement that it is not yet clear how to handle data from mRNA diagnosis in a clinically meaningful way. On the other hand, the greatest oppor- tunities in gene diagnosis lie in this area.

REFERENCES 1 CONNER, B. J., REYES, A. A., MORIN, C., ITAKURA, K., TEPLITZ, R. C. & WALLACE, R. B. (1983). Detection of sickle cell Ps- globin allele by hybridization with synthetic oligonucleotides. Proc. Natl . Acad. Sci. U S A

2 LANGUR, P. R., WALDROP, A. A. & WARD, D. C. (1981). Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl . Acad. Sci.

3 RENZ, M. & KURZ, C. (1984). A colori- metric method for DNA hybridization. Nucl. Acids. Res. 12, 3435-3444. 4 GILLESPIE, D. & SPIEGELMAN, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829-842. 5 NYGAARD, A. P. & HALL, B. D. (1963). A method for the detection of RNA-DNA complexes. Biochem. Biophys. Rex Comm.

6 GALL, J. G. & PARDUE, M. L. (1969). The formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U S A 63, 378-383. 7 GRUNSTEIN, M. & HOGNW, D. S. (1975). Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. U S A 12,

8 SCOTTO, J., HADCHOVEL, M., HERY, C.,

80, 278-282.

U S A 78,6633-6637.

12, 98-104.

3961-3965.

R N A

D N A

Patient Patient Dacs Schm

Fig. 4. Abl oncogene expression in 2 CMLpatients treated with IFN. B, before IFN; A , after IFN. Mononuclear blood cells were purified from blood and divided into two aliquots, one for mRNA immobilization and one for DNA immobilization. Samples taken for mRNA immobilization were handled as described previously.'z In brief, they were deproteinized for 30min at 37°C with 200 pgglml ofproteinase K, exposed to 0.5% BrU 35 and 0.5% sodium desoxycholate, made 12.2 molal in NaI, diluted serially in 12.2 molal NaI and filtered at room temperature through nitrocellulose. Samples taken for DNA immobilization were also processed as described;', they were deproteinized for 30 min at 37 "C with 200 pglml of proieinase K, made 12.2molal in Nal, diluted serially into 12.2 molal NaI, heated to 90-100 "C for 10 min and filtered while hot through nitrocellulose. Twenty- four copies were made of each mRNAIDNA filter. Filters were washed three times in H,O, three times in 70% ethanoll30% H,O then soaked for 10 min in 0.25% aceiic anhydride.l8 Filters were cut into strips each containing one mRNA f DNA copy, thoroughly air-dried and stored in the cold in Ziplock plasiic bags (Dow Chemical Co.). For molecular hybridization, filters were shaken at 42 "C for I7 h in a modified Denhardt's solution.'2 This solution was replaced with hybridization solution containing 50% formamide, SXSSC, 0. I % SDS, 0.05 M-NaPO,, pH 7.0, and about lo6 cpmlml of azP-labeled abl oncogene probe. Afier hybridization, filters were washed as describef12 then radioautographed for I7 h at - 70 "C.

YVART, J., TIOLLAIS, P. & BRECHOT, C. (1983). Detection of Hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 3, 279-284. 9 VOGELSTEIN, B. & GILLESPIE, D. (1979). Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. U S A 76,615-619. 1 0 BRESSER, J. & GILLFSPIE, D. (1983). Quantitative binding of covalently closed circular DNA to nitrocellulose in NaI. Anal. Biochem. 129, 357-364. 11 HAMAGUCHI, K. & GEIDUSCHEK, E. P. (1962). The effect of electrolytes on the stability of the deoxyribonucleate helix. J. Amer. Chem. SOC. 84, 1329-1338. 1 2 BRESSER, J., DOERING, J. & GILLESPIE, D. (1983). Quick-blot: selective mRNA or DNA immobilization from whole cells.

13 GILLESPIE, D. & BRESSER, J. (1983). D N A 2,243-254.


WHAT'S NEW

mRNA immobilization in NaI : Quick-blots. heavy chain variable region gene segment by Biotechniques 1. 184-192. using a screening technique that detects 14 LIE-INJO, L. E., BALASEGARAM, M., mRNA sequencesinwholecelllysates. Proc. LOPEZ, G. G. & HERRERA, A. B. (1983). Natl. Acad. Sci. USA 81, 2410-2414. Hepatitis B virus DNA in liver and whole 16 MEINKOTH, J. & WAHL, G. (1984). blood cells of patients with hepatoma. D N A Hybridization of nucleic acids immobilized 2, 301-308. on solid supports. Anal. Biochem. 138,

DAVID GILLESPIE, M A R Y JO CARANFA, and JOEL BRESSER are in the Department of Hemato'ogy~oncology~

~ ~ ~ ~ ~ ~ $ i ~ ~ f ~ ~ ~ ~ Isd;bz,

Compromise U .S. Pharmaceutical Legislation Enacted Peter Barton Hutt

-

introduction

On September 24, 1984, the Drug Price Competition and Patent Term Resto- ration Act of 1984 was enacted into Law in the United States. This legislation caps 15 years of controversy about the procedures to be used by the U.S. Food and Drug Administration (FDA) in approving the marketing of generic drugs, and about appropriate incentives for developing important new drugs in light of deteriorating patent protection resulting from lengthy regulatory requirements. The bill represents a compromise between these two competing considerations. Title I deals with FDA approval of generic versions of pioneer new drugs previously approved by the agency. Title I1 deals with patent term restoration for new human drug pro- ducts and also for medical devices, color additives, and food additives.

The legislation applies to all drugs marketed in the United States and to all patents granted in the United States, regardless whether the drug is imported or the new drug application (NDA) is owned abroad or the patent is owned abroad. The new statute is lengthy, detailed, and complex, and this article therefore only summarizes the pertinent background and major provisions.

Title I - Generic Drugs

Under the Federal Food, Drug, and Cosmetic Act (FD&C Act) as it was enacted in 1938, FDA was required to review NDAs for all new drugs, to determine whether they had been ade- quately tested for safety. The 1938 Act was amended by the Drug Amendments of 1962 to require that FDA affirmatively

approve all new drugs for both safety and effectiveness.

Drugs which have never been marketed before are called pioneer drugs, and drugs which copy pioneer drugs are called generic drugs. Between 1938 and 1962 some 4000 pioneer drugs had been marketed under NDAs after FDA had reviewed the evidence for their safety. The 1962 Amendments required FDA to review all of those drugs again to determine whether they had also been demonstrated to be effective. Based upon this drug efficacy study implemen- tation (DESI) program, FDA published periodic notices in the Federal Register announcing when a particular pre- 1962 pioneer drug had been reviewed and found to be effective as well as safe. Following publication of those public notices, moreover, the agency approved abbreviated NDAs for generic versions of the pioneer drug, on the basis of data submitted by the generic manufacturer showing that the generic version was bioavailable and bioequivalent to the pioneer drug. An abbreviated NDA for a generic drug was not required to demonstrate the safety and effectiveness of the drug, only that it was equivalent to the pioneer drug that FDA had already found to be safe and effective. Thus, FDA established by administrative procedure, without new legislation, an abbreviated NDA procedure for pre-1962 drugs which facilitated the marketing of generic versions of pioneer drugs.

For the past 15 years, FDA has also considered the possibility of establishing a similar abbreviated NDA system for generic versions of drugs approved by the agency since 1962. Legal and practical considerations have made this

far more complicated, however, and both administrative and legislative ap- proaches have failed up until this time.

FDA did initiate one approach to facilitating the marketing of generic versions of post- 1962 pioneer drugs when it authorized so-called 'paper NDAs' in 1980. A paper NDA must contain all of the animal and human safety and effectiveness information required for a pioneer NDA, but it can be in the form of published literature reporting studies conducted by others, rather than original studies conducted by or for the applicant. The courts upheld the legality of paper NDAs for post-1962 drugs, but because published literature to support the safety and effectiveness for most post-I962 pioneer drugs is not available this procedure has been of limited utility to the generic drug industry.

During the past year, pressure for resolution of the conditions under which generic versions could be approved for post-1962 pioneer drugs intensified from three different sources. First, FDA prepared, and sent forward to the Department of HHS for con- sideration, a proposed administrative regulation with procedures for handling the matter. Second, the generic drug industry brought suit to compel FDA to begin to approve abbreviated NDAs for generic versions of post-1962 pioneer drugs on the same basis as has been used for pre-1962 drugs. Third, Congress- man Waxman introduced legislation and pursued it vigorously. As a result, this legislation was enacted.

The new statute keeps abbreviated NDAs and paper NDAs separate, but applies the exact same rules to both. Because it is easier to obtain an

what's new: the role of chaotropic salts in two-phase gene diagnosis

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