Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Oagurtsov 2011

DNA Cloning
Construction of cDNA Libraries

The main steps in constructing a cDNA library of phage X from a mixture of cellular mRNAs are shown in Figure 91.

To construct a cDNA library, one must first isolate the mRNA of interest from specific Cells of a given tissue.

It is relatively straightforward to separate mRNA from rRNA and tRNA—which are present in cells in much larger quantities—by exploiting the fact that mRNA (unlike rRNA and tRNA) features a poly(A) tail. To separate the mRNA, filters with attached oligo(dT) stretches (short segments of thymidylates) are used.

To synthesize DNA strands complementary to the mRNA starting from a poly(dТ) primer (Figure 91, step 2), researchers use the enzyme Reverse Transcriptase, which was originally discovered in Retroviruses (Section 7.4.2).

The resulting mRNA-cDNA hybrid molecules are converted in several steps into double-stranded cDNA molecules corresponding to all the mRNA molecules present in the initial mixture (steps 3–5).

Each such cDNA molecule possesses an (oligo-dC)•(oligo-dG) stretch at one end and an (oligo-dA)-(oligo-dT) stretch at the other (Figure 91, step 5).

To protect the cDNA from Cleavage by restriction Enzymes, cDNA methylation is performed (Figure 91, step 6).

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Figure 91 - Construction of a cDNA library using bacteriophage λ

Next, in preparation for cloning, short double-stranded DNA molecules containing restriction sites for a specific restriction enzyme are attached to both ends of the cDNA using T4 DNA ligase (Figure 91, step 7). The figure illustrates the attachment of DNA segments containing an EcoRI restriction site.

As mentioned previously, T4 DNA ligase is capable of joining blunt ends of double-stranded DNA molecules that lack sticky ends.

The DNA molecules obtained in this manner are then treated with the appropriate restriction enzyme (EcoRI in the figure). This yields cDNA molecules with sticky ends at both extremities (Figure 91, step 8a).

Simultaneously, "vector arms" are prepared from bacteriophage λ genomes by Digestion with the same restriction enzyme (EcoRI in our example). These vector arms possess sticky ends and contain all the genes necessary for lytic viral growth (Figure 91, step 8(b)). Typically, the insertion region—which is non-essential for library construction (Figure 89, hatched region)—is removed; this is also referred to as the replacement region (Figure 91, hatched region in step 8(b)).

The pool of cDNA and vector arms bearing identical sticky ends is mixed and covalently joined using DNA ligases (Figure 91, step 9). Each resulting recombinant DNA molecule contains the cDNA inserted between the two vector arms of the λ DNA vector.

Next, virions containing the recombinant DNAs are assembled in vitro (as shown in Figure 90) (Figure 91, step 10). Only molecules whose length does not exceed 49 kb can be packaged into the virion.

Finally, the recombinant λ phages are plated on an E. coli lawn to grow individual plaques (Figure 91, step 11).

Because each plaque originates from a single recombinant phage, all progeny λ phages within that plaque are genetically identical and form a clone containing the cDNA derived from a single mRNA molecule. The complete collection of clones from the various plaques constitutes the λ cDNA library.

One characteristic feature of such cDNA libraries stems from the fact that different Genes are transcribed at varying rates (intensities). Consequently, cDNA clones corresponding to highly transcribed genes will be represented multiple times within the library, whereas slowly transcribed genes will be rare or entirely absent.

This property can be advantageously exploited when investigating genes that are transcribed at high rates. Specifically, the "enrichment" of the DNA library with copies of such genes facilitates the screening process.

However, to ensure that the library also contains clones corresponding to slowly transcribed genes, mammalian cDNA libraries must comprise 10⁶–10⁷ (up to ten million) individual recombinant λ phage clones.

Conclusions

For DNA cloning, recombinant DNA molecules are constructed in vitro by inserting DNA fragments into DNA vectors. These recombinant molecules are subsequently introduced into a host Cell, where they replicate to produce A large number of DNA copies.

Restriction enzymes (endonucleases) typically cleave DNA at specific sites 4–8 Base Pairs in length that exhibit a palindromic Structure, thereby generating distinct DNA fragments with complementary single-stranded tails (sticky ends).

Two restriction fragments with complementary ends can be joined together using DNA ligase.

E. coli Plasmid Vectors are circular DNA molecules possessing three essential functional regions: (1) an origin of Replication, (2) an Antibiotic Resistance Gene, and (3) a site where a DNA fragment can be integrated into the plasmid. Transformed cells carrying these vectors form colonies on selective media.

λ phage-based cloning vectors are generated by replacing a non-essential region of the λ genome with a DNA fragment of up to 25 kb in length, followed by the assembly of the resulting recombinant DNA with phage heads and tails pre-synthesized in vitro.

During cDNA cloning, functional mRNA is transcribed into complementary DNA. Following a series of conversions, the single-stranded cDNA is converted into double-stranded cDNA, which is then inserted into a λ phage vector.

A cDNA library is a collection of cDNA clones derived from mRNA extracted from a specific tissue type. A genomic library is a collection of clones containing restriction fragments generated by fragmenting the entire genome.

The number of clones in a genomic or cDNA library must be sufficiently large to ensure that any given starting nucleotide sequence is represented in at least one of the clones.

Self-Assessment Questions

1. What is DNA cloning?

2. What is recombinant DNA?

3. WHAT IS A genetic vector?

4. Why are restriction enzymes used in Recombinant DNA technology?

5. Why are DNA ligases used in recombinant DNA technology?

6. What is a palindromic nucleotide sequence?

7. What region of DNA is called a restriction site?

8. What is the function of the modifying enzyme in the bacterial restriction-modification system?

9. What are "sticky" ends of DNA? How are they generated, and how do they differ from "blunt" ends?

10. What are "blunt" ends of DNA? How are they generated, and how do they differ from "sticky" ends?

11. What are restriction fragments of DNA?

12. What are Plasmids?

13. List the essential components of a plasmid cloning vector.

14. What is a polylinker, and what is its purpose in the design of plasmid vectors?

15. What is The process of cellular genetic transformation, and what are transformed cells?

16. What are the advantages of using bacteriophage λ as a cloning vector compared to plasmid cloning vectors?

17. What is a bacteriophage λ concatemeric DNA, and what is The Role of the Nu1 and A Proteins in concatemer Processing?

18. What is a genomic library?

19. What is complementary DNA, and how is it synthesized?

20. What is the advantage of a complementary DNA (cDNA) library over a standard genomic library?

21. What is the purpose of the enzyme reverse transcriptase in the creation of a complementary DNA (cDNA) library?