Learn about cDNA cloning.
A cDNA (short for complemantary DNA or copy DNA) is a DNA copy of an RNA, usually an mRNA. Sometimes we want to make a cDNA library, a set of clones reprensenting as many as possible of the mRNAs in a given cell type at a given time. Such libraries can contain tens of thousands of different clones. Other times, we want to make one particular cDNA- aclone containing a DNA copy of just one mRNA. This technique we use depends in part on which of these goals we wish to achieve.
If we want to build a cDNA library, we can use a simple but effective strategy that relies on a process called nick translation. The essence of nick translation is the simultaneous removal of DNA ahead of a nick (a single-stranded DNA break) and synthesis of DNA behind the nick. The net result is to move, or translate the nick in the 5'-3' direction. The enzyme we usually use the nick translation is E.coli DNA polymerase I, which has a 5'-3' exonuclease activity that allows the enzyme to degrade DNA ahead of the nick as it moves along.
The central part any cDNA cloning procedure is synthesis of the cDNA from an mRNA template using reverse transcriptase. Reverse transcriptase is like any other DNA-synthesizing enzyme is that it cannot initiate DNA synthesis without a primer. To get around this problem, we take advantage of the poly(A) tail at the 3'-end of most eukaryotic mRNAs and use oligo(dT) as the primer. The oligo(dT) is complementary to poly(A), so it binds to the poly(A) at the 3'-end of the mRNA and primes DNA synthesis, using the mRNA as a template.
After the mRNA has been copied, yielding a single-stranded DNA (the "first strand"), we partially degrade the mRNA with ribonuclease H (RNase H). This enzyme degrades the RNA strand of an RNA/DNA hybrid- just what we need to begin to digest the RNA from our first strand cDNA. The remaining RNA fragments serve as primers for making the "second strand," using the first as the template. This is the nick-translation phase of the process and again, E. coli DNA polymerase I is the enzyme we use to carry out the nick-translation reaction. The net result is a double-stranded cDNA with a small fragment of RNA at the 5'-end of the second strand.
Our next task is to ligate the cDNA to a vector. This was easy with our pieces of genomic DNA cleaved with restriction enzymes, but cDNAs have no sticky ends. We can ligate blunt ends together, even though that is a relatively inefficient process. However, if we want the efficient ligation afforded by sticky ends, we can tack sticky ends (oligo[dC]) onto the cDNA, using an enzyme called terminal deoxynucleotidyl transferase (TdT) or simply terminal transferase and one of the deoxribonucleoside triphosphates. In the case, we use dCTP. The enzyme adds dCMPs, one at a time, to the 3'-ends of the cDNA. In the same way, we attach oligo(dG) ends to our vector and allow the oligo (dC)s to anneal to the oligo(dG)s. This brings the vector and cDNA together in a recombinant DNA that can be used directly for transformation. The base pairing between the oligonucleotide tails is strong enough that no ligation is required before transformation. The DNA ligase inside the transformed cells finally performs the ligation. display_block('cdna'); ?>
See the DNA Molecule in 3-Dimensions