Recombinant cloning

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. 

From the DNA sequence of the resilin recombinant clone, the amino acid sequence of recombinant resilin can be deduced as... 

Figure 4.6. Recombinational cloning (RC). A. Cloning of PCR products using the attB + attP > attL + attR reaction catalyzed by Int and IHF. The result is an Entry Clone that can be used to create functional vectors. B. Conversion of an Entry Clone to a functional vector using the attL + attR > attB + attP reaction catalyzed by Int, Xis and IHF. A wide variety of functional vectors can be constructed by using a Destination Vector with the appropriate promoters and tags.

Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Pick colonies for plasmid miniprepping as usual (if blue-white screening is used then blue colonies should constitute <10% if the reactions were successful. (The blue colonies are derived from inefficiently linearized parental plasmid and should not be picked as they are non- recombinant ). Two colonies are normally sufficient to find a recombinant clone but more may be required. 

WaUiout, A. J., Temple, G. E, Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S. and Vidal, M. (2000). GATEWAY recombinational cloning application to the cloning of large numbers of open reading frames or ORFeomes. Method Enzymol. 328, 575-92. 

The floxed stop cassette of the integrated construct can be deleted in ES cells by transient transfection with a Cre-expressing plasmid (available, e.g., at http //www.addgene. org/) and subsequent low-density plating as selection for recombined clones is impossible. After electroporation, we plate 500-2,000 cells on feeders in a 10 cm dish and analyze 24 clones for recombination by PCR or Southern blot. At least two recombined clones should be analyzed for knockdown efficiency. 

Khalil, A.M., Julius, J.A. and Bachant, J. (2007) One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning. Nucleic Acids Res. 35, el04. 

You just isolated a novel recombinant clone and purified the desired insert (a 10,000 bp linear duplex DNA) from the vector. Now you wish to map the recognition sequences for restriction endonucleases A and B. You cleave the DNA with these enzymes and fractionate the digestion products according to size by agarose gel electrophoresis. Comparison of the pattern of DNA fragments with marker DNAs of known sizes yields the following results ... 

Tedious and time-consuming screening of the recombinant clones The expression levels are influenced by the "positioning effect" and the gene copy number... 

In the shotgun sequencing procedure picking recombinant clones is a random procedure and it is often convenient, having sequenced from one end of a single-stranded insert, to turn the sequence around to be able to sequence from the other end. This is done using the clone tum-around procedure described by Winter... 

RNA probes, and the resulting film exposures can be used to locate the recombinant X plaque responsible for a specific hybridization signal. After a series of plaque purification steps (repeated plating and plaque isolation), an individual X recombinant clone can be purified to homogeneity. 

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