Recombinant clones are cells that have successfully incorporated a piece of foreign DNA, often called the “gene of interest,” into their genetic material. This process is a fundamental aspect of biotechnology and genetic engineering, allowing scientists to study genes, produce proteins, or create genetically modified organisms. Screening is the systematic process of identifying these specific cells from a larger population, ensuring only those with the desired genetic modification are isolated for further use.
The Necessity of Screening
Introducing foreign DNA into host cells is not always a perfect process; not every cell successfully incorporates the genetic material. DNA transformation efficiency rarely reaches 100%. This means that after a transformation, the resulting cell population is a mixture. Some cells will carry the desired gene, while others may not have taken up any DNA at all.
Some cells might also take up a non-recombinant vector, which is the original DNA carrier that recircularized without the foreign DNA insert. Without effective screening, it would be impossible to distinguish cells containing the desired recombinant DNA from those that received no DNA or an undesirable version. Screening is thus essential to identify and isolate the specific cells needed for downstream applications.
Underlying Principles of Screening
The identification of recombinant clones relies on two distinct but related concepts: selection and screening. Selection is typically the first step, eliminating host cells that have not taken up any plasmid DNA. This is often achieved by incorporating an antibiotic resistance gene into the plasmid, allowing only cells that have successfully received a plasmid to grow in the presence of that antibiotic.
Once cells containing any plasmid are selected, screening then differentiates between those that carry the desired recombinant plasmid (with the inserted DNA) and those that contain a non-recombinant, or “empty,” plasmid. This distinction is often made by disrupting a gene on the plasmid when foreign DNA is inserted, or by using a reporter gene whose activity changes upon successful recombination. These mechanisms, such as the disruption of the lacZ gene in blue-white screening, form the basis for visually or functionally identifying recombinant cells.
Common Screening Techniques
Antibiotic Selection
Antibiotic selection ensures that only cells that have taken up a plasmid survive and grow. Plasmids commonly carry genes like ampR (ampicillin resistance) or kanR (kanamycin resistance), allowing host cells to grow on media supplemented with the respective antibiotic. This initial step effectively removes untransformed cells from the population.
Blue-White Screening
Blue-white screening visually differentiates recombinant colonies from non-recombinant ones. This technique relies on the lacZ gene, which encodes the enzyme beta-galactosidase. When foreign DNA is inserted into the multiple cloning site within the lacZ gene, it disrupts the gene’s function, preventing the production of a functional enzyme. In the presence of a substrate called X-gal, functional beta-galactosidase produces a blue color. Therefore, colonies with a non-recombinant plasmid appear blue, while those with a recombinant plasmid (where lacZ is disrupted) remain white.
Colony Polymerase Chain Reaction (PCR)
Colony Polymerase Chain Reaction (PCR) directly detects the inserted DNA fragment without prior DNA extraction. A small portion of a bacterial colony is added to a PCR reaction mixture. Primers designed to bind to regions flanking the insertion site amplify the target DNA. The presence and size of the amplified DNA fragment can then be analyzed using gel electrophoresis, confirming the successful insertion of the desired gene.
Fluorescent Protein Markers
Fluorescent protein markers, such as Green Fluorescent Protein (GFP), provide another visual method for screening. Genes encoding these proteins can be incorporated into the vector so their expression is linked to the successful insertion or expression of the foreign DNA. For example, if foreign DNA is inserted correctly, it might lead to GFP production, causing recombinant cells to glow under specific light, allowing for easy identification.
Verifying Recombinant Clones
After initial screening identifies potential recombinant clones, further verification confirms the presence and correct orientation of the inserted DNA. One common follow-up step involves isolating plasmid DNA from the presumed recombinant clones. This purified plasmid DNA is then subjected to restriction enzyme digest analysis. Specific restriction enzymes cut the plasmid at known sites, and the resulting DNA fragments are separated by size using agarose gel electrophoresis. The pattern of these fragments is compared to the expected pattern of a correctly assembled recombinant plasmid, confirming the insert’s presence and size.
A more precise method for verification is direct DNA sequencing of the cloned insert. Plasmid DNA is isolated, and sequencing primers read the exact nucleotide sequence of the inserted DNA. This accurate method confirms the identity, integrity, and correct orientation of the inserted gene.