Question

What are genomic cDNA libraries and how can they be prepared? What is their advantage?

Answer 1

cDNA or complementary DNA has a sequence opposite to the DNA molecule. It is generally mRNA molecules that have complementary sequences to DNA.

Complete answer: The collection of mRNA segments cloned into separate vector molecules is called the cDNA library.
Complementary DNA or cDNA libraries are a collection of mRNAs that are able to actively synthesize proteins. These mRNA molecules are used to copy them into cDNAs that have complementary sequences to them. Thus, the isolated segments resemble one of the strands of the target DNA. This complementary strand can be made double-stranded and then cloned.
The cDNA libraries can be prepared by the following steps:
The first step is the extraction of mRNA from the eukaryotic cells. The mRNA molecule is first isolated from a eukaryotic cell and is purified using trizol extraction or column purification. The mRNA is then eluted using an elution buffer.
Now, the third step is the construction of cDNA. There are various methods for cDNA construction.
The simplest method is by using the RNase method. In this method, a complementary DNA strand is made using reverse transcriptase to make an RNA-DNA duplex. The RNA strand is later replaced by DNA. This is done using the RNase H enzyme. The left DNA strand then acts as a template and the second strand is synthesized by using DNA polymerase II enzyme.
This is followed by making copies of the newly synthesized DNA. This is done using PCR or polymerase chain reaction.
After the amplification of DNA of interest, the process forwards to the third step which is cloning of the cDNA. The blunt-ended cDNA molecules are attached to the vector in this step. This is done by ligation or by the addition of linker molecules. This completes the construction of cDNA libraries.
Advantages of making cDNA libraries are:
1. A cDNA library is enriched with fragments from actively transcribing genes.
2. There are no introns in cDNA and thus no special eukaryotic transcription machinery is needed for mRNA synthesis in prokaryotic cells.
3. Novel genes can be discovered using cDNA libraries.
4. Gene functioning study can be done on cDNA molecules.

Note: There are certain disadvantages of cDNA libraries. It consists only of sequences that are present in mature mRNA. Sequences that are altered after transcription are not present in cDNA molecules. Also, cDNA molecules represent only those genes that are expressed in the tissue from where the RNA is isolated.