Biotechnology and Bioinformatics (1+1)

2.1.6. cDNA cloning

cDNA cloning plays a major role in current molecular biology. Construct ion of a c DNA library is a highly sophisticated technology that involves a series of enzymatic reactions. The quality and integrity of a cDNA library greatly influence the success or failure in the isolation of the cDNAs of interest.

• If eukaryotic gene is to be clone d and expressed in prokaryotic cell, then directly cutting the source DNA into suitable fragments alone will not be sufficient.
• The difference in the gene organization of eukaryotes and prokaryotes is important.
• Intron s the segments of noncoding sequence s are present in eukaryotic gene. Theses introns are transcribed into mRNA. Such precursor mRNA in eukaryotic cell undergoes post- transcription al modification and removal of introns occurs to give rise to processed mRNA.
• Processed mRNA then gives rise to protein product. In fact there are also post-translational changes occurring in eukaryotic cell. Thus, to get expression in the form of protein product introns have to be removed.
• Bacteria or yeasts do not have necessary splicing mechanism for removal of introns. Hence eukaryotic gene if directly cloned in bacteria or yeast will give rise to precursor mRNA but not the protein product at end.
• This difficulty can be overcome by cDNA route.

The general principles begin with a mRNA that is transcribed into the first-strand DNA, called a complementary DNA or cDNA, which is based on nucleotide bases complementary to the mRNA template.

• This step is catalysed by AMV reverse transcriptase using oligo(dT) primer s.
• The second-strand DNA is copied from the first- strand cDNA using DNA polymerase I, thus producing a double-stranded cDNA molecule.
• Subsequently, the double-stranded cDNA is ligated to an adapter and then to an appropriate vector via T4 DNA ligase .
• The recombinant vector-cDNA molecules are then packaged in vitro and cloned in a specific host, generating a cDNA library.
• Specific cDNA clones can be “fished” out by screening the library with a specific probe .

The messenger RNA is isolated from an appropriate tissues. For example, to obtain the cDNA of growth hormone (GH) gene, pituitary should be used for mRNA preparation, since it is the place where GH is synthesized.

• Taking advantage of the poly–A tail at the 3¹ end of most mRNAs, mRNA is isolated from total cellular RNA by selective binding to and elution from oligo-dT cellulose or poly–U sepharose column.
• The oligo (dT) is complementary to poly (A), so it binds to the poly (A) at the 3¹ -end of the mRNA and primes DNA synthesis, using the mRNA as the template.
• By using reverse transcriptase enzyme, cDNA can be synthesized from mRNA.

After the mRNA has been copied, ssDNA (the “first strand”) is formed and the mRNA is removed with alkali or ribonuclease H (Rnase H). This enzyme degrades the RNA part of RNA/DNA hybrid - remove the RNA from first strand of cDNA.

Next second DNA strand is made using the first as template. We need primer oligo (dc) tail at 3¹ end of the first strand is build, using the enzyme terminal transferase and one of the deoxyribonucleoside triphosphates (dCTP). The enzyme adds dCs, one at a time, to the 3¹ end of the first strand. To this tail, a short oligo (dG) is hybridized, which primes SS syntheses.

DNA polymerase called klenow fragment is used. The klenow fragment contain the DNA polymerase activity and the 3¹ → 5¹ exonuclease activity, but it lacks the 5¹ → 3¹ exon uclease activity normally associated with DNA polymerase I.

Once a double stranded DNA is produced, it is ligated to a vector. Sticky end s are made since cDNA lack sticky ends. To solve this oligo (dc) is added on to cDNA using terminal transferase and dCTP. In the same way oligo (dG) is attached to the ends of vector and allowed the oligo (dC)s to anneal to the oligo (dG)s. This brings the vector and cDNA together in a rDNA that can be used directly for transformation . Plasmid vectors can be used.

Gene libraries of chum salmon, rainbow trout, common carp, grass carp and tilapia were already constructed and available.

There are three steps that are critical for success or failure in the construction of a cDNA library.

• First is the purity and integrity of the mRNAs used for the synthesis of the first-strand cDNAs. Any degradation or absence of specific mRNAs will result in partial-length cDNAs or complete loss of the specific cDNAs, especially for some rare mRNAs.
• The second important step is to obtain full-length cDNAs. If this procedure is not performed well, even if one has a very good mRNA source, the cDNA library is not so good. In that case, one may “fish” out only partial-length cDNAs or no positive clones at all. Once double-strand cDNAs are obtained, they are much more stable as compared with mRNAs.
• A third essential step in cDNA cloning is the ligation of cDNAs with adaptors to vectors. If the ligation fails or is of low efficiency, in vitro packaging of recombinant λDNAs cannot be carried out effectively. In order to construct an excellent cDNA library, elimination of RNase contamination must be carried out whenever possible.

Two major strategies are described for the construction and screening of cDNA library.

• One is subtracted cDNA library in which cDNAs are derived from mRNAs expressed in a specific cell or tissue type but not in another type. The cell/tissue type-specific cDNA clones are greatly enriched in the library, which allows one to readily isolate specific cDNAs copied from rare mRNAs.
• The other is the complete expression cDNA library that includes all cDNA clones from all mRNAs in a specific cell/tissue type.