Cloning screening

Source Vector Total size of DNA screened Number of positive clones Number of clones screened Reference... 

In the directed evolution study, epPCR at various mutation rates was applied, and the total number of clones screened was about 10 000. Of the positive hits identified, some were (R)-, others (iS)-selective. Eight of them were sequenced (Table I) (103). Of particular interest is mutant 1-K2-F5, characterized by a single mutation F432S, because it leads to reversal of enantioselectivity (79% ee in favor of (S)-53). 

Three other forms of the Na+/H+ exchanger have subsequently been cloned. Screening of a rabbit kidney proximal tubule library with an NHE-1 probe (using low stringency hybridization) has led to the identification of two other forms of Na+/H+ exchanger (Tse et al., 1992) that are entirely (NHE-3) or predominantly (NHE-2) expressed in the intestine and kidney. A fourth form of Na+/H+ exchanger (NHE-4) has been identified in the gastrointestinal tract (Orlowski et al., 1992). These isoforms are less sensitive than NHE-1 to amiloride derivatives. 

Probes labeled directly with HRP have been used in many membrane hybridization applications (5,6), including Southern blots. Northern blots, colony and plaque screening, PCR product detection/ identification, YAC clone screening, and RFLP analysis. 

The use of probes directly labeled with horseradish peroxidase (HRP) in conjunction with enhanced chemiluminescence (ECL) allows a flexible approach to hybridizations and detections. This is shown by the diversity of applications in which this labeling and detection system has been used. The applications include Southern blots (1,2) and Northern blots (3), colony and plaque screening for positive clones (4), YAC clone screening (5), and PCR product detection (6,7). 

For expression of the atpB gene, clones screened or enriched for a functional promotor were grown to 0.7 OD6oo and induced by addition of 0.4 mM IPTG. Time dependent... 

Spread the entire transformation over 12 x 150-mm plates of CM(-leu-his-trp+3AT). To determine the number of clones screened, dilute a small aliquot of the transformation 1.10 and 1 100 and plate 50 pL of each dilution on duplicate 100-mm plates of CM(-leu-trp). This protocol generally is sufficient to screen 250,000-300,000 individual clones. One can readily scale up the procedure to screen more of the library in a single experiment. 

O Sullivan, D.J. and Klaenhammer, T.R. (1993). High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 137, 227-231. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

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