Double stranded human globin cDNA was synthesized by use of viral reverse transcriptase from globin mRNA of cord blood of premature infants requiring exchange transfusions. The cDNA was introduced into plasmids and the recombinant DNA plasmids used to transform E. coli X1776. A number of transformants were obtained. Plasmid DNA from selected colonies was isolated and characterized for the type of globin cDNA it contained by three types of procedures: 1) hybridization to previously characterized 3H-labeled a,B and y cDNA; 2) analysis of the size and nature of fragments produced by digestion of the plasmid DNA by different restriction endonucleases; and 3) by rapid DNA sequence analysis of selected DNA fragments produced by restriction endonuclease digestion. Analysis by these techniques of plasmid DNA from different colonies has definitively identified the presence of human a, a or y cDNA sequences in different plasmids.

Several reports have been published on the use of polymorphisms found in the human hemoglobin genes as a means for prenatal diagnosis of sickle cell anemia. The disadvantages ofthis approach reside in its limited application and the need for family analysis. Here we report that, by use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures, a direct analysis can be made. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5'-specific 13-globin gene probe. Sickle cell trait individuals (AS) exhibit an additional band (376 base pairs). Individuals with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. We interpret these changes in banding pattern to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because an analysis can be performed on as little as 20 jig of cellular DNA, the application to prenatal diagnosis of sickle cell anemia should be possible.Recombinant DNA techniques coupled with blot hybridization analysis have proven to be valuable tools for studying the molecular basis of hemoglobinopathies. Various researchers have used blot hybridization to confirm that 8,f3thalassemia (1-5) and hereditary persistence of fetal hemoglobin (1)(2)(3)(4)(5)(6) are the result of gene deletions, whereas a-thalassemia (7-9) and 13-thalassemia (10-12) are due to both gene deletions and point mutations. One study has shown that at least one case of 8-thalassemia is probably due to a base mutation (13). These studies have also been extended to the clinical setting as methods for prenatal diagnosis of various genetic hematological conditions (14-19). Kan and Dozy have reported (20) the finding of a polymorphism for a Hpa I restriction endonuclease site in American Blacks 3' to the ,-globin gene, which was shown to have a 60% association with the sickle cell allele. From their studies, they estimated that blot hybridization using this polymorphism alone could be successfully used for prenatal diagnosis of a sickle cell anemia in 36% of couples at risk. Phillips et al. (21) have combined the Hpa I analysis with a second polymorphism found in the y-globin genes (22). In so doing, they have reported (21) an extension of blot hybridization for prenatal diagnosis of sickle cell anemia to over 80% of the couples at risk. However, both require family studies in order to establish the association ofthe polymorphic sites with the sickle cell allele. This limited application is a major disadvantage of these procedures.A direct analysis of the sickle cell anemia should be possible by use of a restriction enzyme whose recognition sequence is created or eliminated by the sickle cell mutation. This approach would not require family studies and should be useful for all couples at risk.Nienhuis has proposed such a direct analysis with restriction endonuclease Mnl I (23). However, efforts in various laboratories have failed t...

We have reported the direct analysis ofthe allele for 13s-globin by using restriction endonuclease Dde I coupled with blot-hybridization analysis. In that report we predicted that a major use of our analysis could be for the prenatal diagnosis of sickle cell anemia. Here we present such an analysis. In addition, this report also describes the use of a new enzyme Mst H, which also can distinguish the Ps allele from the normal 3-globin allele.

This study examines the effect of different levels of fetal hemoglobin (Hb F) and the presence or absence of genes for alpha-thalassemia on the red cell indices and degree of anemia among 102 patients with homozygous sickle cell disease (S/S) between the ages of 15 and 62 years. Patients were divided into those with an average Hb F of less than 10 gm/L ("low" Hb F group) and those with greater than 10 gm/L ("high" Hb F group). alpha-Thalassemia was assessed by restriction enzyme analysis of DNA by the Southern blotting technique. Homozygosity for the beta(s) gene was confirmed by restriction enzyme analysis of DNA using the enzyme Mst II. There were 51 patients with four alpha-globin genes, 28 of whom had "high" and 23 "low" Hb F levels. Fifty-one patients had alpha-thalassemia, 38 of whom were heterozygous and 13 homozygous for the 3.7 kb alpha-thalassemia deletion. Nine had "high" and 31 had "low" Hb F. Irrespective of alpha-globin genotype, patients in the high Hb F group had a higher mean Hb, Hct, MCV, and MCH than those in the low HB F group. In patients without alpha-thalassemia Hb F was positively correlated with MCV and MCH (p less than 0.001), patients with high Hb F levels having macrocytosis confirmed by microhematocrit studies. Patients with alpha-thalassemia had a lower MCHC than patients with four alpha-globin genes and this was not significantly affected by the level of Hb F. The combination of alpha-thalassemia and high levels of Hb F appears to result in a distinctive S/S phenotype that is similar to the type of S/S disease described in Southern India.

The sequence of most of the human beta globin messenger RNA and large sections of the alpha globin messenger RNA has been determined. Partly because of genetic polymorphism, it was necessary to clone globin complementary DNA in order to extend the analysis. Purified human fetal globin messenger RNA was isolated and used as a template by reverse transcriptase to produce duplex complementary DNA molecules. These molecules were linked in vitro to plasmid DNA by use of T4 ligase in the presence of Escherichia coli Pol 1. Several colonies transformed by these molecules have been shown to hybridize with labeled human globin complementary RNA.