cDNA library  
 
     
  Formation of a cDNA library  
     

 

cDNA Library /¦Ë phage library

cDNA is created from mRNA with the use of an enzyme known as reverse transcriptase. In eukaryotic cells, a tRNA and rRNA. Extracted RNA content from a cell can be passed through a chromatography columnon which a strand of complementary nucleotides ; such as short uracil [poly(U)] or thymidine extacts (oligo-dT) are attached to a matrix in the column. The mRNA's poly-(A) tail can bind to the oligo-DTs, whilst the rest is washed away. Elution with low-salt buffer releases the mRNA from the oligo-dTs.

Once the purified mRNA is recovered, reverse transcriptase is used to make templates of double-stranded DNA from the mRNA templates. The newly formed DNA templates are then inserted into bacteria plasmids using restriction enzymes (to cut open the plasmid) and ligase (to seal up the ends once the DNA has annealed into the plasmid).

 

 
 

Unfortunately, a very low percentage of plasmids actually have the DNA successfully inserted into the plasmid despite a high percentage cut by restriction enzymes. Many plasmids simply remain cut open or anneal with itself again. Therefore, to isolate those plasmids that do have the DNA successfully inserted, an extra antibiotic resistance gene is usually inserted along with the DNA. The bacteria is then grown in a medium with this antibiotic. All bacteria with plasmids that have successfully taken in the DNA (and hence the antibiotic resistance), will then survive, with all the others dying off.

The surviving colonies are then grown to reproduce the plasmid with the DNA, which is then purified. This is also why these types of libraries are called cDNA or complementary DNA, because it is a complemetary strand of DNA to the mRNA in question.

The ¦Ë phage library is similar with cDNA library in technology and have its own strong point and poor point. The ¦Ë phage library has some merits of great capacitance, high quality and representative character , and it can be stored in a long time. But the operation is very fussy and the efficiency is very low. Furthermore , the screening techniques are limited . On the basis of it ¦Ë TriplEx2 can be transformed into plasmid pTriplEx2, we can transform the ¦Ë phage vectors into the plasmid vectors by random samples from the outcome of plasmids and plasmid PCR. All these showed that the method can be widely used to study the function genornics.