Inverse PCR (CAT#: STEM-MB-0191-WXH)

Name: Inverse PCR
SKU: STEM-MB-0191-WXH
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Introduction

Inverse polymerase chain reaction (Inverse PCR) is a variant of PCR that is used to amplify DNA with only one known sequence. One limitation of conventional PCR is that it requires primers complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers may be designed.
Inverse PCR is especially useful for the determination of insert locations.

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Principle Applications Procedure Materials

Principle

Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites

Applications

• Determination of insert locations.
• Identification of unknown flanking regions.
• Identification of unknown mutations such as gene rearrangements, gene fusion oncogenic gene arrangement on a chromosome.
• Insertion of viral gene segments or plasmid.
• Transposable element studies, identification and characterization.
• Site-directed mutagenesis.

Procedure

1.Identification of known DNA region having flanking unknown DNA sequence.
2.Restriction digestion of gDNA.
3.Ligation of digested unknow DNA fragments.
4.Amplification of ligated circular DNA molecule.
5.Sequencing of the unknown DNA region.