ABSTRACT
Inverse polymerase chain reaction (iPCR) is a powerful tool that can be used for the sequence analysis of DNA when only one end of a DNA sequence is known. iPCR1-3 amplies DNA from a circular template using primers with their 3′ ends directed away from each other (Figure 21.1). Cloning and sequencing PCR products are facilitated by the use of vectors, for example, pCR®2.1 (Invitrogen), which contain a thymine overhang. iPCR is very useful for supplementing conventional PCR screens that often yield only a substantial fragment of the gene of interest. An iPCR strategy used in conjunction with commercial cloning vectors and modern sequencing technology can rapidly yield accurate DNA sequencing results for several kilobases (kB) and allows one to “walk” both upstream and downstream of the known DNA sequence to obtain additional sequence. The methodology facilitates amplication of unknown DNA anking sequences without the labor involved in constructing and screening libraries. Identication of anking sequences on either side of a known sequence can be challenging. Other PCR-based methods for gene isolation, including random priming or the use of adapter sequences (anchored and rapid amplication of cDNA ends (RACE) PCR), employ a single sequence-specic primer, consensus or degenerate primer, or randomly prime off the template. These approaches frequently yield a low signal-to-noise ratio. iPCR requires two sequence-specic primers and generally yields a high signal-to-noise ratio. In addition, the iPCR primer pair can be used to amplify multiple circularized gene fragments that contain overlapping restriction sites. However, the iPCR technique requires circularization of DNA fragments within an optimum size range (see Section 21.4).
