A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allelespecific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell g3-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.Sickle cell anemia is the prototype ofa genetic disease caused by a single base-pair mutation, an A -* T transversion in the sequence encoding codon 6 of the human f3-globin gene. In homozygous sickle cell anemia, the substitution of a single amino acid (Glu -* Val) in the B-globin subunit of hemoglobin results in a reduced solubility of the deoxyhemoglobin molecule and erythrocytes assume irregular shapes. The sickled erythrocytes become trapped in the microcirculation and cause damage to multiple organs.Kan and Dozy (1) were the first to describe the diagnosis of sickle cell anemia in the DNA of affected individuals based on the linkage of the sickle cell allele to an Hpa I restriction fragment length polymorphism. Later, it was shown that the mutation itself affected the cleavage site of both Dde I and Mst II and could be detected directly by restriction enzyme cleavage (2, 3). Conner et al. (4) described a more general approach to the direct detection of single nucleotide variation by the use of allele-specific oligonucleotide hybridization. In this method, a short synthetic oligonucleotide probe specific for one allele only hybridizes to that allele and not to others under appropriate conditions. All of the above approaches are technically challenging, require a reasonably large amount of DNA, and are not very rapid. The polymerase chain reaction (PCR) developed by Saiki et al. (5) provided a method to rapidly amplify small amounts of a particular target DNA. The amplified DNA could then be readily analyzed for the presence of DNA sequence variation (e.g., the sickle cell mutation) by allelespecific oligonucleotide hybridization (6), restriction enzyme cleavage (5, 7), ligation of oligonucleotide pairs (8, 9), or ligation amplificatio...

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.

A method is described for analyzing the association of the opiate narcotic levorphanol with brain tissue into three components: nonsaturable, saturable nonspecific, and saturable stereospecific. The method may be of general applicability for the study of the interaction of drugs with body tissues. In mouse brain the stereospecific binding of levorphanol represents only 2% of the total association of drug with tissue, and it was found only in certain membrane fractions. The material responsible for the stereospecific binding might be the opiate receptor.

We describe a restriction gene (Akvr-1, for AKR virus restriction) that is polymorphic for two alleles, Akvr-1 R (restrictive) and Akvr-1 r (susceptible), in a feral population of mice (Mus musculus domesticus) at a squab farm near Lake Casitas (LC) in southern California. Akvr-1" is a dominant allele that exhibits 100% penetrance in prevention of viremia of AKR endogenous retrovirus and of virus-mediated lymphoma in LC (Akvr-l RR) X AKR F1 hybrids. The restriction phenotype segregates as a single Mendelian locus in backcrosses to AKR mice. Akvr-1 R likewise is effective in restriction of NB-tropic Moloney murine leukemia virus-induced viremia and NB-tropic Friend virus-induced splenomegaly but fails to restrict expression or pathogenesis of LC-derived amphotropic retrovirus.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.