Human globin messenger RNA: importance of cloning for structural analysis

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The complete sequences of human beta globin messenger RNA (mRNA) and significant portions of alpha globin mRNA were previously known.
• Genetic polymorphism presented challenges for extending the analysis of globin gene sequences.

Purpose of the Study:

• To extend the sequence analysis of human globin genes.
• To overcome limitations posed by genetic polymorphism in globin mRNA sequencing.

Main Methods:

• Isolation of purified human fetal globin mRNA.
• Synthesis of duplex complementary DNA (cDNA) using reverse transcriptase.
• In vitro ligation of cDNA to plasmid DNA using T4 ligase and Escherichia coli DNA polymerase I.
• Transformation of bacterial colonies with recombinant plasmids.
• Hybridization of transformed colonies with labeled globin cDNA probes.

Main Results:

• Successful cloning of human globin complementary DNA (cDNA).
• Generation of recombinant plasmids containing globin cDNA sequences.
• Identification of transformed bacterial colonies that hybridized with labeled globin cDNA, confirming successful cloning.

Conclusions:

• Cloning globin cDNA is an effective strategy to extend sequence analysis, particularly in the presence of genetic polymorphism.
• This approach facilitates detailed study of globin gene structure and function.