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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber

Lecture 27

cDNA Libraries

Isolation of corresponding genetic information

Instead of synthesizing a desired gene, can we used the amino acid information to directly isolate the corresponding genetic information?

  1. Genomic DNA
  2. mRNA

If we are considering genomic DNA from eukaryotes, then there are a couple of things to consider:

  1. The coding region for a gene of interest may be interrupted by one or more intron regions, and thus the complete coding region could be quite long.
  2. To a first approximation, it does not matter which tissue we use to isolate the genomic information, i.e. the genomic content is the same in all tissues.

If we are considering mRNA from eukaryotes, we may realize the following advantages:

  1. Introns will be spliced out and the mRNA will contain a contiguous coding region.
  2. Tissue specific expression of the protein of interest may allow us to isolate appropriate mRNA at enhanced levels, i.e. in tissues where the protein is expressed the mRNA levels are considerably higher than the corresponding genomic levels (there are many more molecules of mRNA than copies of the gene).


A "library" is a convenient storage mechanism of genetic information.

cDNA library construction

"Reverse transcription" is a mechanism whereby genetic information contained in mRNA is converted back into a double stranded DNA form.

The enzyme responsible for this is an RNA dependent DNA polymerase called reverse transcriptase.

MMLV will use mRNA as a template, but requires a primer (it can extend a DNA primer but cannot synthesize one).

Therefore we can use poly dT as a single primer for a variety of different eukaryotic mRNA's.

Note that we have produced complementary DNA (or cDNA) to the original mRNA strand.

If we can introduce "nicks" into the RNA half of this DNA/RNA duplex then the situation would be very similar to that observed in "lagging strand" synthesis of prokaryotic genomic DNA.

Note that we will potentially have either a residual 5' RNA cap region, or a gap at the 5' end of the original mRNA strand.

Insertion of cDNA into plasmid.

To complete our construction of a useful cDNA library we need a way to maintain and propagate our cDNA.

  1. Homopolymeric tailing
  2. Linker addition

Homopolymeric tailing

Terminal transferase is an unusual DNA polymerase found only in a type of eukaryotic cell called a prelymphocyte.

How can we insert this into a plasmid?

  1. The Pst I recognition sequence and cleavage site is
    5' C T G C A G 3'
    3' G A C G T C 5'
  2. Cleavage of this site by Pst I, followed by G-tailing will produce
    5' C T G C A (G)n G 3'
    3' G (G)n A C G T C 5'

Thus, the Pst I recognition cleavage site is regenerated and the C-tailed cDNA insert can be excised again from the library vector by cleaving with Pst I.


An alternate method to insert cDNA fragments into a library vector is through the addition of "linkers".

The steps in linker addition are as follows:

  1. Treatment of cDNA with S1 nuclease (to remove possible 5' cap mRNA fragment remaining in cDNA duplex
  2. Convert potential "ragged" ends to blunt by treatment with Pol I (will fill in 5' overhangs and chew back 3' overhangs)
  3. Methylate cDNA at potential internal Eco RI sites by treatment with Eco RI methylase (plus S-adenosyl methionine)
  4. Ligate linkers to blunt, methylated cDNA using T4 DNA ligase
  5. Cut linkers with Eco RI restriction endonuclease
  6. Remove linker fragments from cDNA fragments by agarose gel electrophoresis
  7. Ligate cDNA to vector DNA fragment (opened up by Eco RI restriction endonuclease

1998 Dr. Michael Blaber