The Polymerase Chain Reaction (PCR) is a foundational technique used to rapidly create millions of copies of a specific, known segment of DNA. Standard PCR requires pre-existing knowledge of the DNA sequences that flank the target region to design primers. Inverse PCR (IPCR) is a specialized variation developed to overcome this limitation. IPCR allows for the amplification and subsequent identification of DNA regions adjacent to a known sequence, even when the flanking sequences themselves are entirely unknown.
How Inverse PCR Differs from Standard PCR
Conventional PCR is an “inward-facing” process, where two primers bind to the outer boundaries of the target region and extend toward each other, amplifying the sequence between them. This requires knowing the precise sequence at both ends of the DNA segment. Inverse PCR, by contrast, is an “outward-facing” process. The difference lies in its ability to amplify unknown flanking sequences when only a small internal region is known. The primers are designed to bind within the known sequence, but they are oriented to extend away from each other, amplifying the adjacent, uncharacterized DNA.
This technique is necessary because outward-facing primers cannot amplify a linear piece of DNA. If the template remains linear, the primers would fall off the ends during the extension step, leading to no amplification. Therefore, the DNA template must undergo a structural modification before amplification can proceed.
Why Circularization is Necessary
The issue of outward-facing primers on a linear template is solved by converting the DNA fragment into a circular molecule. Circularization brings the unknown flanking regions into close proximity, placing them between the binding sites of the inverted primers. This process begins by treating the genomic DNA with a restriction enzyme, which cuts the DNA at specific recognition sites outside of the known sequence. The enzyme is chosen to generate a manageable fragment size, typically a few kilobases.
Next, the linear fragments undergo a ligation reaction using DNA ligase. Under conditions favoring intramolecular ligation, the two ends of a single DNA fragment are joined, forming a closed DNA loop or circle. This circular structure transforms the DNA segment into a continuous loop, enabling inverse amplification.
The Step-by-Step Mechanism of Inverse PCR
The inverse PCR method involves four primary stages:
Preparation and Digestion
The target DNA undergoes digestion using a restriction enzyme that does not cut within the known sequence. This generates linear fragments where the known region remains intact and the unknown flanking sequences are positioned at the ends.
Ligation and Circularization
DNA ligase is introduced under dilute conditions to promote the self-joining of the fragment’s two ends. This molecular fusion creates a closed, circular DNA template. In this structure, the unknown flanking sequences become contiguous, and the known internal sequence is continuous with the flanking DNA.
Primer Design
This stage involves creating two primers that bind to opposite strands within the known sequence. Unlike standard PCR primers, these are oriented to extend outward from the known sequence, across the newly formed ligation junction. The circular template allows these outward-facing primers to proceed with synthesis around the loop.
Amplification
The circularized DNA is used as the template in a standard thermal cycling process. During the extension phase, the outward-facing primers synthesize new DNA that continues through the unknown flanking region, crosses the ligation point, and completes the circle. This process generates a linear DNA product consisting of the entire unknown flanking region, which can then be sequenced.
Key Research Applications
Inverse PCR is a technique for identifying the precise locations of mobile genetic elements, such as transposons or viral DNA, that have inserted themselves into a host genome. Researchers use the known sequence of the inserted element to design the outward-facing primers, allowing them to amplify and sequence the adjacent host DNA. This reveals the exact genomic insertion site, which aids in understanding the element’s effect on gene function.
The technique is also used in “genome walking,” which systematically sequences adjacent, uncharacterized regions of a chromosome. When only a partial gene sequence is known, IPCR allows scientists to amplify upstream or downstream regulatory elements, or to fully characterize the entire gene structure. IPCR also finds utility in site-directed mutagenesis, where the entire circular plasmid is amplified to introduce a specific change in the DNA sequence.