Lecture 2: Review of Recombinant DNA Technology (contd.)

Construction of DNA libraries

A repository of a large number of recombinants comprises what is called a DNA library. The generation of DNA libraries is one of the most important steps of recombinant DNA technology. DNA libraries are a collection of DNA fragments from an organism that can be cloned into any of the vectors mentioned in Lecture 1.

An important concept to review before talking about the construction and screening of DNA libraries is how a DNA molecule is converted into a protein molecule. The scheme given below summarizes the steps from a DNA to a protein molecule in a eukaryotic organism.

A more detailed look at the process of transcription is depicted below (taken from the web).

Before we get into how the different libraries are constructed, we have to realize the need for constructing these libraries. In the Biotechnology industry, a lot revolves around expressing proteins of interest, be it in the agricultural, biomedical, or pharmaceutical fields. To be able to express a protein of interest on a large scale, the gene encoding that protein has to be isolated, cloned into an appropriate expression vector and then expressed into protein. To fish out a gene from the genome of any organism GENE LIBRARIES are constructed. The different systems available for protein expression will be discussed later.

Libraries can be generated in any one of the following ways.

Chopping up the entire DNA content of an organism either mechanically or with restriction enzymes to generate small fragments (Genomic DNA libraries) .
Changing the DNA into mRNA (process called transcription) and using the mRNA population thus generated to make the library (cDNA libraries).

A genomic library

A) Represents every gene essentially equally
B) Has overlapping clones
C) Has promoters, introns etc.
D) Most clones contain only part of the coding sequence of a gene

Advantage of cDNA libraries is that if the gene of interest is highly expressed in a particular tissue there will be abundance of that mRNA and it will be easy to isolate because it will be enriched in a cDNA library made from that particular tissue. A cDNA library will represent individual genes, although not all genes will be represented. Also no promoters or introns will be present. A schematic of the construction of the 2 libraries is shown below:

Differences between a cDNA and genomic library are also given here

There are several modifications and variations of cDNA libraries that are mentioned below. An overview of the cDNA library construction, its variants and screening is given in this article.

Full-length cDNA libraries: their advantage over conventional cDNA libraries and the construction strategy is explained here with illustrations.
PCR-mediated cDNA libraries: A PCR-directed cDNA library is an approach to analyze cell-specific gene expression where the amount of tissue is limiting. In most cDNA libraries the first step is the isolation of total RNA followed by removal of the highly abundant rRNA and tRNA components to isolate mRNA. However in PCR approach to cDNA synthesis, total RNA is the starting material.

Schematic for construction of PCR-mediated cDNA library

Subtractive cDNA library: a powerful method for the enrichment of genes expressed highly under a specific condition .
Expression cDNA library: to express the protein of interest from the corresponding gene. This involves making the libraries in vectors that will express the protein encoded by the inserted cDNA sequence. Prokaryotic expression libraries can be constructed in either plasmid or bacteriophage l vectors that carry a strong promoter and a ribosome-binding site. The insert sequence can be expressed by itself as a protein, or as a fusion protein in which it is expressed attached to another protein. Expression libraries can be constructed in eukaryotic organisms such as yeast.

Screening of DNA libraries

The purpose of screening the DNA libraries is to identify a gene (clone) of interest. Once a genomic or cDNA library is available, it can be used for isolation of a gene sequence.

There are several methods of screening libraries for clones of interest:

Hybridization: Before talking about the various approaches used to identify a specific clone/colony/gene, let’s understand the meaning of hybridization as commonly used in biotechnology.

A DNA molecule exists as a double helical molecule with two polynucleotide chains that are held together by base pairing between the complementary base pairs i.e. A::T and G::C. An aqueous solution of DNA when heated at a high temperature ³ 94 0C or exposed to alkaline pH ³ 13, results in the disruption of the complementary base pairs that normally hold the two strands of DNA double helix together. For many years this process, called DNA denaturation was thought to be irreversible. In 1961 , it was discovered that complementary single strands of DNA will readily reform the double helix if the temperature is brought down to ~ 65 0C. This process is called renaturation or hybridization . This process can occur between any two single-stranded nucleic acid chains (DNA/DNA; DNA/RNA; RNA/RNA) as long as they have a complementary nucleotide sequence.

This is the most commonly used and reliable method of screening libraries for the gene of interest. It has several features that make it a popular approach.

Large number of clones (recombinants carrying inserts) can be analyzed simultaneously
The cDNA clones need not be full-length
Does not require that an antigenically or biologically active product be synthesized in the host cell
Will detect all clones that contain cDNA sequences, unlike antibodies, which will detect only those clones in which the cDNA has been inserted into the vector in the correct reading frame.

The steps involved in the screening by hybridization are detailed on p71-72 of Glick & Pasternak.

Genetic selection or complementation screening: This technique can be used only when mutations are available in the gene of interest, because the selection is based on the ability of a gene to restore the normal phenotype (appearance) to a mutant cell. For example looking for a gene that codes for 'penicillin'. Take cells that are known 'penicillin mutants' so that there is a mutation in the gene encoding for penicillin, and the normal functional product is not obtained. Transform with recombinant DNA clones in the library and plate them out on medium containing penicillin. Only clones or cells that have the pen r gene will be able to grow in the presence of penicillin.

The expression libraries are screened for the presence of a specific gene by

Immunological Assay : Antibodies (if available) can be used to detect cDNA clones that express one or more antigenic determinants. Membranes with the library colonies are treated with a lysis solution to break open the cells. The membranes are then incubated with a solution containing the antibody from a desired protein. Additionally, labeled double-stranded oligonucleotide probes can be used to screen for recombinant proteins that bind specific DNA sequences, or labeled protein probes to identify recombinant proteins that specifically bind to the protein probe.
Determination of protein expression : If the target gene codes for an enzyme, then the library can be screened for the function of an enzyme to identify the cells that are producing the biologically active form of that enzyme.

Once the putative (most likely) clone of choice has been isolated, it has to be confirmed that it is the correct clone. This can be done by:

Expressing the clone (producing functionally active form of the protein encoded by the gene in the clone of interest) in prokaryotic or eukaryotic cells to check that it displays the correct biological or enzymatic activity.

Check the correspondence between portions of the nucleotide sequence of the cDNA and the amino acid sequences of peptides derived from the purified protein.
Immunoprecipitation of the peptides synthesized in vitro or in vivo from transcripts of the cDNA clone by antibodies raised against the protein of interest.

Some other methods based on the principle of hybridization:

Southern Blot Hybridization: Published in 1975 by E M Southern, this process enables investigators to identify the locations of genes and other DNA sequences on restriction fragments separated by gel electrophoresis. The essential feature is transfer of the DNA molecules that have been separated by gel electrophoresis onto a specially treated membrane (paper). Such transfers of DNA to membranes are called Southern blots(named after the person who developed this technique). For the sake of complementarity, the transfer of RNA to membranes is called Northern blots. The transferred DNA is first immobilized or bound permanently on the membrane by UV irradiation followed by incubation with a labeled DNA probe to detect specific sequences.
FISH (Fluorescent In Situ Hybridization): described well on this site or this.
PCR: discussed last week.