The Polymerase Chain Reaction (PCR) can be used to rapidly generate DNA fragments for cloning, provided that a suitable source of template DNA exists and sufficient sequence information is known to permit design of primers specific for the desired amplicon. Unlike traditional cloning, PCR offers the ability to readily clone DNA fragments that may be of low abundance in a complex sample such as genomic DNA, or cDNAs that correspond to rare mRNA transcripts. PCR products can be digested and ligated by traditional means, ligated directly (blunt or TA ends), or used in ligation independent cloning (LIC) or seamless cloning applications, such as Gibson Assembly® or NEBuilder HIFI DNA Assembly (NEBuilderhifi.com).
During a typical PCR, template DNA (containing the region of interest) is mixed with deoxynucleotides (dNTPs), a DNA polymerase and primers. Primers are short segments of complementary DNA that base-pair with the template DNA, upstream of the region of interest, and serve as recruitment sites for the polymerase. PCR involves a series of temperature cycles that are controlled automatically by the use of a thermocycler that precisely controls both the reaction temperature and the duration of each temperature step, ensuring efficient amplification (for more details about PCR, see DNA Amplification section).