Sources of DNA for Cloning

By using the genetic code, one can take the amino acid sequence of a given protein and come up with a series of trinucleotide codons that would direct that protein s synthesis. Then, thanks to advances in nucleic acid chemistry, the DNA with those codons in the proper sequence can readily be made in a laboratory. In short, one can synthesize an artificial gene. 

FIGURE 3.11 Construction of a synthetic gene from chemically synthesized oligonucleotides. 

Another method is to extract the DNA, then treat it with an enzyme that cleaves double-stranded DNA at specific sequences (a restriction enzyme see below). By partially digesting the DNA, large fragments can be generated moreover, these fragments will contain identical overlapping sequences at their ends that will help later in the cloning process. 

A third alternative starts with an extract of RNA, not DNA. Mature eukaryotic mRNA contains a long run or tail of adenine residues at its 3 end. The poly(rA) tail can be hybridized with an oligomer of thymine residues, and the oligo(dT) can then be used as a primer for a particular kind of DNA polymerase known as reverse transcriptase. This enzyme, a polymerase associated with retroviruses, will use RNA as a template to make a complementary DNA copy of the RNA, creating a DNA-RNA double-stranded hybrid. In another round of synthesis, the enzyme can replace the RNA strand entirely with DNA, so that the RNA-DNA hybrid is completely converted to double-stranded DNA containing an exact copy of the original RNA sequence. This DNA molecule is known as cDNA because it has a strand that is complementary to (or a copy of) the original RNA. 

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