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PCR

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).

For routine, robust PCR reactions OneTaq DNA Polymerase is the most common choice of enzyme. This polymerase leaves predominantly template-independent single adenines (A) at the 3’ end of the PCR product. For high-fidelity PCR, a proofreading DNA polymerase should be used. Such enzymes do not create single base overhangs, leaving blunt termini. A consideration of the ends of PCR products, including their phosphorylation status, is important to subsequent cloning strategies (see End Modification). When PCR primers include restriction enzyme sites the PCR products can be digested and ligated by traditional means. 

Vector molecules for cloning may also be produced by PCR. Restriction sites included in the primers allow generation of sticky ends (single strand overhangs) to facilitate cloning of restriction fragments. Otherwise, a blunt ended vector can be produced by PCR using a high-fidelity proofreading polymerase or by blunting of the single base 3’ overhang produced by Taq polymerase. Reverse transcription of RNA to first strand complementary DNA (cDNA) followed by PCR (RT-PCR) allows cloning of double-stranded DNA molecules that correspond to the gene transcripts (for mRNA, see the cDNA synthesis).

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