It is hard to imagine areas of biological research that have not been touched by molecular cloning. Cloning involves insertion of genetic sequences in mostly bacterial vectors such as plasmids and allows researchers to obtain large number of copies of the inserted sequence. Once cloned, sequences can be readily manipulated and expressed, thereby facilitating functional studies.
Traditional methods for cloning DNA fragments involve the ligation of restriction digest fragments obtained from chromosomes, plasmids, or cosmids into specific sites on a selected cloning vector. Standard molecular cloning techniques require enzymatic modification and purification steps to prepare DNA sequences for ligation and transformation into bacterial cells. Typically, the first step in the transfer of a DNA sequence for cloning involves digestion
of the DNA by restriction enzymes to produce terminal sequence overhangs, also termed cohesive ends, that facilitate ligation into a similarly prepared vector with compatible cohesive ends. Following each digestion step, the released adapter sequences need to be removed so they will not compete with the desired insert during subsequent ligation and significantly increase the number of spurious recombinants. DNA electrophoresis followed by recovery of the desired DNA fragments from the gel is the most frequently used purification method for separating adapters from the desired product.
While restriction digest is still important in the cloning process, aspects of its use have changed. Rather than solely relying on restriction digestion to obtain fragments for cloning, currently the polymerase chain reaction (PCR) often is employed to generate and prepare sequences for cloning. DNA or cDNA resulting from reverse transcription of RNA can serve as templates for PCR amplification. One of the main advantages of PCR is that it provides a straight forward way to introduce restriction sites necessary for directional cloning of the amplified sequence by incorporating them in the amplification primers.
Standard techniques often involve multiple purification steps, each associated with significant product losses, therefore requiring large amounts of starting DNA to guarantee sufficient vector and insert material to complete subsequent ligation and transformation steps. Because of the numerous applications that require production of recombinant DNA molecules, a rapid method is needed for transferring DNA fragments from a PCR product or other source into the desired vector.
In this paper, we describe the use of these ultrafiltration devices in various steps of the cloning workflow that can provide a more streamlined process and compare them against traditional methods of subcloning PCR-generated fragments.