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).
FAQs for PCR
Protocols for PCR
- Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)
- Control Reaction Protocol for PreCR Repair Mix
- Loop-mediated Isothermal Amplification (LAMP)
- Luna® Universal One-Step RT-qPCR Kit Protocol (E3005)
- Luna® Universal Probe One-Step RT-qPCR Kit Protocol (E3006)
- Luna® Universal Probe qPCR Master Mix Protocol (M3004)
- Luna® Universal qPCR Master Mix Protocol (#M3003)
- Sequential Reaction Protocol for PreCR Repair Mix
- Standard Reaction Protocol for PreCR Repair Mix
Anatomy of a Polymerase - How Structure Effects Function
Polymerase Fidelity: What is it, and what does it mean for your PCR?
Understanding Variability in DNA Amplification Reactions
Molecular Cloning Technical Guide
Download the latest Molecular Cloning Technical Guide for help with product selection, protocols, tips for optimization and trouble-shooting.
The PCR brochure provides product information on the wide range of DNA polymerases available from NEB, including tools for selection and troubleshooting tips.
Reagents & Tools for Molecular Cloning brochure
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- DNA Polymerase Selection Chart
- PCR Troubleshooting Guide
- Taq PCR Kit Troubleshooting Guide
- General Guidelines for Successful RNA Purification Using the Monarch Total RNA Miniprep Kit
- Guidance on Choosing Sample Input Amounts when Using the Monarch Total RNA Miniprep Kit
- Guidelines for PCR Optimization with OneTaq® and OneTaq® Hot Start DNA Polymerases
- Guidelines for PCR Optimization with Taq DNA Polymerase
- Guidelines for PCR Optimization with Thermophilic DNA Polymerases
Other Tools & Resources
Make sure you're using the optimal polymerase for your DNA amplifications. Get tips on choosing the right DNA Polymerase for your application.
Not sure why Q5® is your best choice for high-fidelity amplification of GC-rich targets? NEB's scientists will show you why we call Q5 an "ultra-high fidelity polymerase".
Here are some quick tips for getting the most out of NEB's Q5® High-Fidelity DNA Polymerase.
Looking for tips on dealing with GC-bias in DNA amplification? NEB scientists have the expertise you need!
>210 of NEB's restriction enzymes are 100% active in a single buffer. Learn more about CutSmart® Buffer and why it matters to you.