Five Clone Screening Methods for Every Scientist

Clone screening is the process of identifying and isolating specific clones that exhibit desired characteristics or contain the target gene of interest from a larger population. It involves testing or analyzing multiple clones to determine which ones have the appropriate genetic modification or gene expression levels. Various techniques can be used for screening, such as polymerase chain reaction (PCR), colony hybridization, reporter gene assays, and enzyme activity assays.

On the other hand, clone selection involves choosing clones from the population of screened clones. The clones are selected based on optimal characteristics such as growth rate, expression levels, enzyme activity, and productivity, in addition to the desired genetic trait.

Clone screening helps verify that your gene of interest was successfully cloned. Pick your favorite method from these 5 ways to verify that your gene of interest was successfully cloned—the classic way, the powerful way, the precise way, the quick way, or the most accurate way.

The plasmid vector used in this method contains a gene for β-galactosidase (lacZ), which is involved in the metabolism of lactose. This lacZ gene has a multiple cloning site (MCS) where a DNA fragment of interest (insert) can be ligated. When the DNA of interest is inserted into the plasmid, the gene is disrupted, and the bacterial colony becomes white in color. The absence of DNA insert in the vector produces blue colonies because the vector does not contain the insert.

The plasmids are introduced into competent E. coli cells through the process of transformation. The transformed E.coli are plated on agar plates containing an antibiotic (to select for bacteria that have taken up the plasmid) and X-gal, a lactose analog that turns blue when hydrolyzed by β-galactosidase substrate for β-galactosidase (Figure 1).

Insert absent: If the plasmid does not have an insert, the lacZ gene remains intact and produces functional β-galactosidase. This enzyme cleaves X-gal, producing a blue product. Thus, colonies containing non-recombinant plasmids will appear blue.

Insert present: If the plasmid has an insert, the lacZ gene is disrupted and does not produce functional β-galactosidase. As a result, X-gal is not cleaved, and these colonies remain white. Colonies containing recombinant plasmids will appear white.

In summary, if DNA insert is present, the colonies will appear white. If the DNA insert is not present, the colonies will appear blue. Since it is possible to obtain false positives, it’s always best to follow up using one of the other methods (PCR, restriction digest, or DNA sequencing).

Using restriction enzymes to check the presence and direction of the insert is a precise and easy method for screening colonies. This approach uses restriction mapping to verify the presence of target DNA insert within the plasmid. Recombinant plasmid DNA is isolated from an overnight culture of transformed bacterial cells. The plasmid DNA from recombinant clones is then digested with restriction enzymes. The plasmid digests are run on an agarose gel to verify that the vector backbone and insert are of the expected sizes (Figure 2).

Colony screening by PCR is well suited for inserts shorter than 3 kb. Some PCR enzyme master mixes allow direct amplification of DNA from intact cells. A portion of an individual colony is directly added to a PCR master mix for amplification. The PCR products can then be analyzed by agarose gel electrophoresis, E-gels, or capillary electrophoresis methods.

The most accurate way to verify recombinant colonies is by Sanger sequencing. Plasmid DNA is first isolated from an overnight bacterial culture. The insert is then identified using sequencing primers appropriate for the selected vector. The entire insert is sequenced and compared with the exact sequence of the insert.