General Principles And Considerations Of An Expression cDna Library

Unlike the subtracted cDNA library described previously, a complete cDNA library theoretically contains all cDNA clones corresponding to all mRNAs expressed in a cell or tissue type. An expression cDNA library refers to one in which all cDNAs are cloned in the sense orientation so that all the cDNA clones in the library can be induced to express their mRNAs and proteins. As a result, this cDNA library can be screened with specific antibodies against the expressed protein of interest or with a specific nucleic acid probe.

The general principles and procedures are similar to those of the subtracted cDNA library described earlier. Briefly, mRNAs are transcribed into the first-stranded DNAs, or called complementary DNA (cDNA), based on nucleotide bases complementary to each other. This step is catalyzed by AMV reverse transcriptase using oligo(dT) adapter-primers. The second-stranded DNA is copied from the first-stranded DNA using RNase H that creates nicks and gaps in the hybridized mRNA template, generating 3'-OH priming sites for DNA synthesis by DNA polymerase I. Following treatment with T4 DNA polymerase to remove any remaining 3' protruding ends, the blunt-ended, double-stranded cDNAs are ready for the ligation with appropriate adaptors or linkers. The resultants are then packaged in vitro and cloned in a specific host, generating a complete express cDNA library. In theory, this type of cDNA library preserves as much of the original cDNA as possible, which can allow "fishing" out any possible cDNA clones by screening the cDNA library as long as a specific probe or specific antibodies are available (Figure 3.6).


EcoR I

EcoR I


AMV Reverse Transcriptase tTTTTT dNTPs, Adapter Primers 42°C, 60 min



Xba I

Xba I

Xba I adapter-primer mRNA 1st cDNA

I RNase H, DNA polymerase I ▼ dNTPs, 14YC, 60-120 min

* -1 * AAAAAfXban 3' Degradation of mRNA

TTTTTT I 5' and synthesis of 2nd I T4 DNA polymerase strand cDNA

| dNTPs

I T4 DNA ligase ▼ EcoR I adaptors/linkers

Xba I

3' 2nd strand cDNA 5' 1st strand cDNA

Digestion with EcoR I and Xba I

; AATTTTtXbaI 53



Xba I

\ DNA ligase, vector arms mRNA


Packaging in vitro


Xba I

A complete expression cDNA library

Screening of the cDNA library

FIGURE 3.6 Construction of a complete expression cDNA library using a directional cloning strategy.

0 0

Post a comment