You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library constructed using the vector shown in Problem 18. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS (the positions of these sequences are shown in the figure in Problem 18). The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors. Can you identify the start of the coding region in the end of the gene? What does the sequence preceding the start codon represent?

Key Concepts

cDNA Cloning

cDNA cloning involves synthesizing complementary DNA (cDNA) from messenger RNA (mRNA) using the enzyme reverse transcriptase. This process allows researchers to create a DNA copy of a specific gene, which can then be inserted into a vector for further study. The directionality of cloning, as indicated by the use of specific restriction sites like EcoRI and XhoI, ensures that the cDNA is inserted in the correct orientation for expression.

Start Codon and Coding Region

The start codon, typically AUG, marks the beginning of the coding region in mRNA, which is the sequence that will be translated into a protein. The coding region is flanked by untranslated regions (UTRs) that play roles in regulation and stability of the mRNA. Identifying the start codon is crucial for understanding where translation begins and for determining the functional protein product of the gene.

Untranslated Regions (UTRs)

Untranslated regions (UTRs) are segments of mRNA that are not translated into protein but are important for the regulation of gene expression. The sequence preceding the start codon is part of the 5' UTR, which can influence mRNA stability, localization, and the efficiency of translation initiation. Understanding UTRs is essential for comprehending how genes are regulated at the post-transcriptional level.