Key

Core known region Unknown genomic DNA I PCR primers

RS1 (2) Restriction site

Fig. 2 Inverse PCR of a linear construct. Schematic for the PCR amplification of an unknown region from a linear construct.

Biotech, Little Chalfont, UK)]. Because a quality DNA starting material is important for efficient IPCR, this should be the first issue that needs to be addressed.

The restriction enzyme required to cleave the target DNA region is crucial in that it must produce a fragment of appropriate length for the subsequent ligation and PCR. Small fragments (<200 bp) prove difficult to circularize, whereas larger fragments (> 3 kb) may be problematic for PCR amplification. However, Benkel and Fong[10] have published a hybrid method allowing amplification of larger fragments by combining long-range (distance) PCR with IPCR, and accordingly labeled it long-range inverse PCR (LR-IPCR). In addition to the fragment size, the positions of the restriction sites are important when deciding what is required from the amplification. If amplification of both flanking regions is sought, then it is essential that the restriction enzyme of choice does not cleave within the known core region. Alternatively, if only amplification of one side of the core region is necessary, the enzyme must cleave inside the core region, but leave enough core sequence to allow the design of a primer pair for PCR.[3,11]

The element of IPCR becomes more difficult when there is no restriction map available of the unknown region. It has been suggested that by using a restriction enzyme with a four-base recognition sequence, enough IPCR suitable fragments would be generated.1-11-1 However, this method could be very random and the use of Southern blots to identify restriction sites is a more controlled option. In all cases, numerous restriction digests should be evaluated to elucidate the most appropriate and efficient for PCR of the unknown region.

If IPCR on a linear construct is to be undertaken, care should be taken regarding the choice of restriction enzyme used to linearize the circular construct. The restriction site has to be within the known region between the 5' ends of the primers, not downstream of this or in the unknown sequence, or the template for subsequent PCR will be destroyed. An alternative to restriction digestion of the circular construct, yet still enhancing it as a PCR template,

V Plasmid

Was this article helpful?

0 0
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment