DNA Preparation

Molecular cloning involves introducing DNA, as an insert, into a vector molecule. The DNA to be cloned can be obtained by cutting it out of a source DNA by digestion with restriction enzymes, by copying it from a source molecule by either the Polymerase Chain Reaction (PCR) or Reverse Transcription-PCR (RT-PCR), or by assembling it from short DNA pieces (oligonucleotides). These methods all require that the DNA source is sufficiently free of contaminants that could potentially inhibit the enzyme activities (endonucleases, polymerases) involved in processing the DNA for cloning.

Traditional cloning by restriction endonuclease digestion can use any of a number of different source DNA types. Genomic DNA can be digested with a restriction enzyme and cloned into a compatible vector site to produce a library of different inserts, all from the same source DNA. DNA already cloned into one vector can be transferred (subcloned) to a new recipient vector by cutting out the DNA with restriction enzymes and cloning into the corresponding sites of the second vector. This is frequently undertaken to facilitate protein expression or transcription of RNA, for example, which might not be possible from the original vector.

PCR is often used for generating DNA for cloning and frequently restriction sites are incorporated into the primer sites so that the amplified DNA can be digested and cloned into compatible restriction sites of the cloning vector. Any type of DNA containing the desired sequence can serve as the template for PCR. Cloning with two distinct restriction enzymes ensures that non-compatible ends are generated on each molecule, thereby preventing simple vector recircularization and forcing inserts to be cloned directionally. This can be important for ensuring a translational open reading frame for protein expression. Modification of DNA ends following restriction digestion can be helpful in certain situations. For example, in non-directional cloning, where a single restriction enzyme is used, dephosphorylation of the digested vector DNA will prevent recircularization of the vector, thereby increasing the proportion of the desired recombinant DNA molecules.

PCR is increasingly used for preparing DNA for cloning applications. Amplified DNA can either be cloned directly, or following restriction digestion with restriction sites engineered into the primers used for PCR. Alternatively, amplified DNA can be used in Seamless Cloning strategies such as NEBuilder HiFi DNA Assembly or in Ligation Independent Cloning. The vector molecules for these cloning methods may also be produced by PCR. DNA amplified with Taq polymerase has a template-independent single adenosine (A) added at the 3’ ends which allows cloning into complementary T-tailed vectors. High-fidelity proofreading polymerases do not add additional bases allowing cloning of the amplified DNA into blunt-ended restriction sites. Depending on the chosen cloning strategy, the ends of amplified DNA can be modified by A-tailing, blunting, or addition or removal of 5’-phosphate groups. Complementary DNA (cDNA) generated by reverse transcription of RNA can also be amplified by PCR. This ability to synthesize DNA from RNA templates enables cloning of sequences corresponding to gene transcripts.

Learn more about the different methods to prepare DNA: Restriction Enzyme Digestion, PCR and Reverse Transcription (cDNA Synthesis)