Target amplification for DNA analysis by the polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard laboratory technique. An enzymatic reaction, as simple to perform as it is satisfying to contemplate, the PCR solves two of the more universal problems in the chemistry of natural nucleic acids. It allows for the physical separation any particular sequence of interest from its context; and then provides for an in vitro amplification of this sequence which is virtually without limit. The surprising robustness of PCR derives from its fortuitous combination of three phenomena, each of which is intrinsically powerful. The first of these is the impressive ability of almost all oligodeoxynucleotides to bind tightly and specifically to their complementary nucleic acid sequences, discriminating easily between hundreds of thousands of sites. The second familiar phenomenon is illustrated by the notion that the probability for the occurrence of a compound action is the product of the individual probabilities for the occurrence of each of its components. The third phenomenon embodied in the polymerase chain reaction relates to the branching structure of its propagation and the inherent robustness attached to such a form. Consideration of the above leads to certain generalities regarding the relative utility of various protocols for carrying out the PCR. Specific conditions of time, temperatures, concentrations, etc. will be described, as well as sample preparation and analytical methods.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection.

Human immunodeficiency virus (HIV) has been associated with acquired immunodeficiency syndrome and related disorders. Assays to detect antibodies to HIV proteins have been developed and used to screen sera for the identification of individuals who have been exposed to the virus. Although these serological tests have significant sensitivity and specificity for detecting exposure to the virus, they do not provide direct identification of HIV. We report here the application of recently developed nucleic acid amplification and oligonucleotide-based detection procedures for the identification of HIV sequences in established infected cell lines and in cells cultured from infected individuals.

Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes.

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A)+ mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR alpha chain, were used to prime a cDNA synthesis reaction on various poly(A)+ mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha mRNA was confirmed by primer extension and blot analysis. The cDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A)+ mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of cDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.

Opiate antagonist receptor binding in vivo: evidence for a new receptor binding model.

The in vivo accumulation and retention of the opiate antagonist tracers [3H]diprenorphine and [3H]naloxone at cerebral opiate receptor sites in rats exceed that expected from their known in vitro receptor affinities. The [3H]diprenorphine serum and brain levels can be stimulated with a pharmacokinetic model that contains the receptors in a micro-compartment. The receptor micro-compartment consists of a population of binding sites next to a diffusion boundary which restricts ligand diffusion away from the receptor. Such an arrangement introduces a delay in the binding equilibrium of potent antagonists with the receptor sites and an increase in the apparent in vivo receptor affinity at subsaturating doses of the ligand; at saturating ligand concentrations these functions of the receptor micro-compartment are abolished. A physiological interpretation of the receptor micro-compartment could be the location of clustered opiate receptor sites on the exterior cell surface next to the synaptic cleft as the diffusion boundary. This kinetic approach involving a combination of pharmacokinetics and drug-receptor interactions permits the quantitative analysis of receptor site availability in the intact animal. Our results support the hypothesis that only one receptor population affects the in vivo disposition of the antagonist tracers, while they do not exclude the presence of low affinity binding sites that have been observed with the use of [3H]naloxone in vitro. Moreover, the binding site population observed in vivo may be responsible for mediating opiate agonist analgesia.

Morphine persistence in rat brain and serum after single doses.

The disposition of morphine in rat brain and serum was determined over 48 hr after subcutaneous doses. Free morphine was measured by a specific assay using 3H-labeling together with high-pressure liquid chromatography separation, with a sensitivity of 1 nM (0.3 ng of morphine per ml). This study revealed the persistence of free morphine in nanomolar concentrations over at least 24 hr after a single analgesic dose. The terminal half-life of morphine elimination was 5 hours. Total radioactivity was retained in the body at much higher concentrations. Similar disposition of [C-1-3H]morphine and [N-14CH3] morphine ruled out any major metabolic alterations at these positions, including N-demethylation. Irreversible binding to insoluble tissue components, which has previously been linked to tolerance, was observed only to the extent of less than 20% of total tissue radioactivity and was not unique to brain tissue. The persistence of morphine and its metabolites may be related to protracted opiate effects such as withdrawal symptoms after addiction.

Angiotensin I-converting enzyme and angiotensin II levels in women receiving an oral contraceptive.

In 11 healthy, normotensive young women taking contraceptive medication (Enovid) for at least one year, plasma levels of angiotensin II were significantly higher than in healthy male and female controls. No significant difference was seen in the serum activity of angiotensin I-converting enzyme measured in vitro. Although serum angiotensin I converting enzyme activity is stimulated in several conditions in which other components of renin-angiotensin-aldosterone system are increased, this is not the case during administration of estrogens.

Prevention of pulmonary vascular changes of chronic alveolar hypoxia by inhibition of angiotensin I-converting enzyme in the rat.

To test the hypothesis that the renin-angiotensin system is involved in the development of the pulmonary vascular patholigic changes of chronic alveolar hypoxia, two groups of rats were exposed to 0.5 atm. for 21 days. One group received SQ 20,881 (2 mg. per kg.) every 8 hours subcutaneously and the second group received normal saline. A third group of rats was maintained at normobaria. Rats receiving SQ 20,881 had significantly less pulmonary arterial hypertrophy and right ventricular hypertrophy than hypobaric animals. SQ 20,881-treated rats showed a significant increase in adrenal weight but a marked reduction in the thickness of the zona glomerulosa, as compared with the other groups. Our findings indicate that angiotensin II is necessary in the development of the pulmonary vascular structural changes of chronic alveolar hypoxia.